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A humanized IgG but not IgM antibody is effective in prophylaxis and therapy of yellow fever infection in an AG129/17D-204 peripheral challenge mouse model
Abstract
Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne virus found in tropical regions of Africa and South America that causes severe hepatic disease and death in humans. Despite the availability of effective vaccines, YFV is responsible for an estimated 200,000 cases and 30,000 deaths annually. There are currently no prophylactic or therapeutic strategies approved for use in human YFV infections. Furthermore, implementation of YFV 17D-204 vaccination campaigns has become problematic due to an increase in reported post-vaccinal adverse events. We have created human/murine chimeric MAbs of a YFV-reactive murine monoclonal antibody (mMAb), 2C9, that was previously shown to protect mice from lethal YFV infection and to have therapeutic activity. The new chimeric (cMAbs) were constructed by fusion of the m2C9 IgG gene variable regions with the constant regions of human IgG and IgM and expressed in Sp2 murine myelomas. The 2C9 cMAbs (2C9-cIgG and 2C9-cIgM) reacted with 17D-204 vaccine strain in an enzyme-linked immunosorbent assay and neutralized virus in vitro similarly to the parent m2C9. Both m2C9 and 2C9-cIgG when administered prophylactically 24h prior to infection protected AG129 mice from peripheral 17D-204 challenge at antibody concentrations ≥1.27 μg/mouse; however, the 2C9-cIgM did not protect even at a dose of 127 μg/mouse. The 17D-204 infection of AG129 mice is otherwise uniformly lethal. While the m2C9 was shown previously to be therapeutically effective in YFV-infected BALB/c mice at day 4 post-infection, the m2C9 and 2C9-cIgG demonstrated therapeutic activity only when administered 1 day post-infection in 17D-204-infected AG129 mice.
Supplementary resources (2)
Nucleotide Sequence
February 2010
Nucleotide Sequence
February 2010
... Epitopes in DII and DIII elicit virus-neutralizing monoclonal antibodies (MAbs) and anti-DIII MAbs usually have more potent in vitro neutralization titers than anti-DII MAbs (Roehrig, 2003). Certain anti-E MAbs possess antiviral prophylactic and therapeutic activity in animal models of flavivirus infection (Brandriss et al., 1986;Engle and Diamond, 2003;Gould et al., 1986;Hawkes et al., 1988;Johnson and Roehrig, 1999;Julander et al., 2014;Kimura-Kuroda and Yasui, 1988;Mathews and Roehrig, 1984;Thibodeaux et al., 2012b), and the humanized anti-West Nile virus (WNV) EDIII MAb MGAWN1 has demonstrated efficacy in animal models and undergone Phase I clinical trials to demonstrate safety in humans (Beigel et al., 2010). We recently developed a YFVtype-specific chimeric murine-human MAb, 2C9-cIgG, and demonstrated its prophylactic and therapeutic activity in two animal models of infection (Julander et al., 2014;Thibodeaux et al., 2012b). ...
... Certain anti-E MAbs possess antiviral prophylactic and therapeutic activity in animal models of flavivirus infection (Brandriss et al., 1986;Engle and Diamond, 2003;Gould et al., 1986;Hawkes et al., 1988;Johnson and Roehrig, 1999;Julander et al., 2014;Kimura-Kuroda and Yasui, 1988;Mathews and Roehrig, 1984;Thibodeaux et al., 2012b), and the humanized anti-West Nile virus (WNV) EDIII MAb MGAWN1 has demonstrated efficacy in animal models and undergone Phase I clinical trials to demonstrate safety in humans (Beigel et al., 2010). We recently developed a YFVtype-specific chimeric murine-human MAb, 2C9-cIgG, and demonstrated its prophylactic and therapeutic activity in two animal models of infection (Julander et al., 2014;Thibodeaux et al., 2012b). MAb 2C9-cIgG reacts with both virulent and vaccine YFV, binding to an epitope in DII of the E protein (Lobigs et al., 1987). ...
... MAb 2C9-cIgG reacts with both virulent and vaccine YFV, binding to an epitope in DII of the E protein (Lobigs et al., 1987). Interferon receptor-deficient AG129 mice were protected from or successfully treated after challenge with 17D-204 when the cMAb was inoculated 24 h prior to or 24 h after viral infection (Thibodeaux et al., 2012b). MAb 2C9-cIgG was more effective in an immunocompetent hamster model challenged with virulent YFV Jimenez strain (Julander et al., 2014). ...
The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.
... Although germline sequences are potentially less immunogenic and therefore preferred in humanization of antibodies, IgG-derived sequences are sometimes more favorable. For example, Thibodeaux et al. (2012) showed that chimeric antibodies constructed by fusing the variable domain of murine antibody 2C9 to the constant region of IgG were effective in prophylaxis and therapy of yellow fever infection, but those with the constant region of IgM were not. They found the Fc region of IgM-humanized 2C9 antibody responsible for this inability. ...
... They found the Fc region of IgM-humanized 2C9 antibody responsible for this inability. This conclusion derived from the fact that these two antibodies, 2C9-cIgM and 2C9-cIgG, had similar variable domains and differed only in Fc portions (Thibodeaux et al., 2012). It has previously been reported that loss of murine IgG Fc region reduces the protection capacity of antibody (Schlesinger & Chapman, 1995). ...
... It has previously been reported that loss of murine IgG Fc region reduces the protection capacity of antibody (Schlesinger & Chapman, 1995). Activity and properties of humanized antibodies also vary depending on the class of IgG (Thibodeaux et al., 2012). Cheung and colleagues found that humanized antibodies with the same variable domain, but with different Fc regions derived from IgG1 or IgG4, exhibited different properties. ...
This article reviews recent advances achieved during recent years on various aspects of antibody humanization theories and techniques. Common methods for producing humanized antibodies including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting, as well as advantages and disadvantages of each of these methods and their applications are discussed.
... Mice typically develop encephalitis when infected with YFV, making them less well-suited as models of human disease. We recently developed a murine model of disease in mouse strain AG129, which is deficient in α/βand γ-interferon receptors, peripherally challenged with YF 17D-204 vaccine (Thibodeaux et al., 2012a) and used this model system for evaluation of 2C9-cIgG (Thibodeaux et al., 2012b). Although AG129 mice lack a functional interferon response, they provide a useful model for initial proof of concept studies. ...
... The MAb was diluted in phosphate-buffered saline (PBS). A hamster dose was determined using results from the AG129 mouse studies (Thibodeaux et al., 2012b), based on surface area conversion. The West Nile virus (WNV)-specific, humanized MAb MGAWN1 was obtained from Macrogenics Inc. (Rockville, MD). ...
... We previously showed that mMAb 2C9 and 2C9-cIgG therapy in YFV-17D-204-infected AG129 mice, which lack interferon-α/β and -γ receptors, led to increases in survival rates only when administered at or before 24 hpi (Thibodeaux et al., 2012b). This immunocompromised mouse strain is highly susceptible to challenge with vaccine strain YFV, which replicates efficiently and causes both viscerotropic disease and CNS infection (Thibodeaux et al., 2012a). ...
Yellow fever virus (YFV) causes significant human disease and mortality in tropical regions of South and Central America and Africa, despite the availability of an effective vaccine. No specific therapy for YF is available. We previously showed that the humanized monoclonal antibody (MAb) 2C9-cIgG provided prophylactic and therapeutic protection from mortality in interferon receptor-deficient strain AG129 mice challenged with YF 17D-204 vaccine. In this study we tested the prophylactic and therapeutic efficacy of this MAb against virulent YFV infection in an immunocompetent hamster model. Intraperitoneal (ip) administration of a single dose of MAb 2C9-cIgG 24 h prior to YFV challenge resulted in significantly improved survival rates in animals treated with 380 or 38 μg of MAb compared to untreated animals. Treatment with the higher dose also resulted in significantly improved weight gain and reductions in serum alanine aminotransferase (ALT) and virus titers in serum and liver. Prophylactic treatment with 2C9-cIgG 24 h prior to virus challenge prevented the development of a virus-neutralizing antibody (vnAb) response in hamsters. Administration of a single ip dose of 380 μg of 2C9-cIgG as late as 72 h post-YFV challenge also resulted in significant improvement in survival rates. Hamsters treated at 4 to 72 h post-virus challenge developed a robust vnAb response. Enhanced survival and improvement of various disease parameters in the hamster model when MAb 2C9-cIgG is administered up to 3 days after virus challenge demonstrate the clinical potential of specific antibody therapy for YF.
... A mAb designated A5 was identified using phage display technology and showed efficacy in an immunodeficient YFV-17D challenge model (23). A humanized mAb designated 2C9 showed benefit in hamsters against the Jimenez strain (24) and in AG129 mice against YFV 17D-204 challenge (25), supporting the proof of principle for antibodies as a medical countermeasure for YFV. Fully human mAbs with native heavy and light chain pairing, however, are preferred for use in human therapy. ...
... Previous studies support the concept that the administration of mAbs may be effective in reducing viral load and disease. The murine DII-specific mAb 2C9 and the murine-human chimeric mAb 2C9-cIgG exhibited therapeutic activity when administered 1 day after infection in interferon alpha/beta/gamma receptor-deficient strain AG129 mice challenged with the YF17D-204 vaccine (25) and against virulent YFV infection in an immunocompetent hamster model (24). A second murine-human chimerized mAb, designated 864-cIgG, recognizes DIII and neutralizes the YFV 17D-204 vaccine substrain but did not protect AG129 mice against 17D-204 infection, probably due to its For panels C to H, a Mann-Whitney test was performed (*, P , 0.05; **, P , 0.01). ...
Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in areas of endemicity. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed the identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting the YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV-17D vaccine strain in vitro. YFV-136 also potently inhibited infection by multiple wild-type YFV strains, in part, at a postattachment step in the virus replication cycle. YFV-136 showed therapeutic protection in two animal models of YFV challenge, including hamsters and immunocompromised mice engrafted with human hepatocytes. These studies define features of the antigenic landscape of the YFV E protein recognized by the human B cell response and identify a therapeutic antibody candidate that inhibits infection and disease caused by highly virulent strains of YFV.
... A mAb designated A5 was identified using phage display technology and showed efficacy in an immunodeficient YFV-17D challenge model (23). A humanized mAb designated 2C9 showed benefit in hamsters against the Jimenez strain (24) and in AG129 mice against YFV 17D-204 challenge (25), supporting the proof of principle for antibodies as a medical countermeasure for YFV. Fully human mAbs with native heavy and light chain pairing, however, are preferred for use in human therapy. ...
... Both neutralizing Previous studies support the concept that the administration of mAbs may be effective in reducing viral load and disease. The murine DII-specific mAb 2C9 and the murine-human chimeric mAb 2C9-cIgG exhibited therapeutic activity when administered 1 day after infection in interferon alpha/beta/gamma receptor-deficient strain AG129 mice challenged with the YF17D-204 vaccine (25) and against virulent YFV infection in an immunocompetent hamster model (24). A second murine-human chimerized mAb, designated 864-cIgG, recognizes DIII and neutralizes the YFV 17D-204 vaccine substrain but did not protect AG129 mice against 17D-204 infection, probably due to its low potency (reported as 10 mg/mL in a 90% plaque reduction neutralization test) (34). ...
Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in endemic areas. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed for identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from the circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV 17D vaccine strain in vitro . YFV-136 also potently inhibited infection by multiple wild-type YFV strains, in part, at a post-attachment step in the virus replication cycle. YFV-136 showed therapeutic protection in two animal models of YFV challenge including hamsters and immunocompromised mice engrafted with human hepatocytes. These studies define features of the antigenic landscape on YFV E protein recognized by the human B cell response and identify a therapeutic antibody candidate that inhibits infection and disease caused by highly virulent strains of YFV.
... Mice and monkeys can be protected by passive immunization with monoclonal antibodies to YF-17D. [60][61][62] The level of neutralizing antibodies is usually measured either by the log neutralization index (LNI), in which the potential of a sera to neutralize virus infectivity is evaluated using a constant serum concentration and increasing amounts of challenge virus, or by a more conventional plaque-reduction neutralization test (PRNT) assay. 63 In animal models, an LNI.0.7 is associated with protection against challenge with a virulent virus. ...
... Importantly, mice can survive experimental YFV infection after passive immunization with humanized IgG. 62 Many studies have been conducted to identify target antigens for these antibody responses. ...
Flaviviruses comprise approximately 70 closely related RNA viruses. These include several mosquito-borne pathogens, such as yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), which can cause significant human diseases and thus are of great medical importance. Vaccines against both YFV and JEV have been used successfully in humans for decades; however, the development of a DENV vaccine has encountered considerable obstacles. Here, we review the protective immune responses elicited by the vaccine against YFV to provide some insights into the development of a protective DENV vaccine.Cellular & Molecular Immunology advance online publication, 5 October 2015; doi:10.1038/cmi.2015.76.
... Given that the peripheral challenge used in these immunodeficient mouse models also generally resulted in longer time to death compared to previous i.c. challenge models using wild-type or vaccine YFV strains, this may also make them more amenable to efficacy studies with candidate therapeutics (Thibodeaux et al., 2012b). ...
... Passive immunotherapy with polyclonal anti-YFV serum was shown to protect against lethal disease in NHPs when antiserum was administered up to 3 days post-challenge with YFV (Davis, 1934;Pettit and Stefanopoulo, 1931), although Davis reported decreased survival rates when treatment occurred more than 24-48 h after infection. Therapy using typespecific monoclonal antibodies (MAbs) targeted against the YFV E or NS1 proteins has also been shown to protect mice and hamsters against lethal disease when administered prior to or shortly after YFV challenge (Brandriss et al., 1986;Julander et al., 2014b;Schlesinger et al., 1985;Thibodeaux et al., 2012b). The emergence of neutralization-resistant variants has been reported in vitro for YFV (e.g. ...
... There is currently no approved treatment for YFV infection. Although there have been numerous other monoclonal antibodies (mAb) isolated from previously infected humans, vaccinated individuals, and various sources, there are now no active clinical trials testing interventional YFV therapeutics (6)(7)(8)(9)(10). Previous clinical trials have included two limited studies. ...
Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks.
... 17D-IRES displayed limited viscerotropism in AG6 mice. Mice deficient in alpha/ beta and gamma interferon (IFN-a/b and -g) receptors have been widely used for viscerotropism testing (25). To assess the viscerotropism of 17D-IRES, groups of AG6 mice were inoculated with 10 4 PFU of 17D-IRES or 17D through the intraperitoneal (i.p.) route. ...
Yellow fever (YF) continually spreads and causes epidemics around the world, posing a great threat to human health. The YF live attenuated vaccine 17D is considered the most efficient vaccine available and helps to successfully control disease epidemics.
... There is currently no approved treatment for YFV infection. Although there have been numerous other monoclonal antibodies (mAb) isolated from previously infected humans, vaccinated individuals, and various sources, there are now no active clinical trials testing interventional YFV therapeutics (6)(7)(8)(9)(10). Previous clinical trials have included two limited studies. ...
Few countermeasures to treat Yellow Fever virus (YFV) infection are under development, because vaccines have helped to limit new infections. Unfortunately, vaccine hesitancy, supply deficits, and a paucity of therapeutic options have left individuals at risk. Here, we tested potent YFV-specific neutralizing monoclonal antibodies in rodents and non-human primates. We administered antibodies during acute pathogenic YFV infection and demonstrate that we can prevent severe disease and death. Given the severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing Yellow Fever cases during outbreaks around the world.
One Sentence Summary
Therapeutic monoclonal antibodies prevent death from YFV infection.
... Given the incredible potential of mAbs as drugs, the last decade has witnessed an explosion in the efforts to characterize mAbs against a variety of pathogenic viruses, including different strains of influenza virus [3,4], human immunodeficiency virus 1 (HIV-1), cytomegalovirus (CMV), hepatitis C virus (HCV), Ebola virus, Marburg virus [5,6], dengue virus [7,8], Hendra and Nipah viruses [9][10][11], yellow fever virus [12,13], and West Nile virus [14]. Remarkably, just in the past year and a half, more than 60 antibodies binding the spike protein of SARS-CoV-2 that have been isolated from COVID-19 patients [1,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] have demonstrated high potency in neutralizing the virus ex vivo and in protecting against virus infection and virus spread post-infection in preclinical models such as mice, hamsters, and nonhuman primates (NHPs) (reviewed in [34]). ...
Since the first approval of monoclonal antibodies by the United States Food and Drug Administration (FDA) in 1986, therapeutic antibodies have become one of the predominant classes of drugs in oncology and immunology. Despite their natural function in contributing to antiviral immunity, antibodies as drugs have only more recently been thought of as tools for combating infectious diseases. Passive immunization, or the delivery of the products of an immune response, offers near-immediate protection, unlike the active immune processes triggered by traditional vaccines, which rely on the time it takes for the host’s immune system to develop an effective defense. This rapid onset of protection is particularly well suited to containing outbreaks of emerging viral diseases. Despite these positive attributes, the high cost associated with antibody manufacture and the need for a cold chain for storage and transport limit their deployment on a global scale, especially in areas with limited resources. The in vivo transfer of nucleic acid-based technologies encoding optimized therapeutic antibodies transform the body into a bioreactor for rapid and sustained production of biologics and hold great promise for circumventing the obstacles faced by the traditional delivery of antibodies. In this review, we provide an overview of the different antibody delivery strategies that are currently being developed, with particular emphasis on in vivo transfection of naked plasmid DNA facilitated by electroporation.
... It has been demonstrated using the inbred AG129 line mice deficient for α and β interferon receptors model that humanized IgG-derived MAbs possess higher preventive and therapeutic efficiency against YFV-caused disease compared to IgM-derived MAbs (Table 1). In particular, chimeric human/mouse MAbs able to protect mice from the lethal YFV infection were developed based on the mouse 2C9 IgG YFV-reactive antibody [33]. ...
... To date, upon YFV infection, there are no specific and effective antiviral reagents available that can be used to treat the disease. Neutralizing antibodies proved to be a promising way to treat viral diseases, including infections with HIV, influenza virus, and other flaviviruses (Rouvinski et al., 2015;Thibodeaux et al., 2012;Wang et al., 2016b;Wu et al., 2015;Zhou et al., 2010). Previously, several neutralizing antibodies targeting the E protein of YFV have been isolated and show potent neutralizing activity against YFV (Daffis et al., 2005). ...
... To date, upon YFV infection, there are no specific and effective antiviral reagents available that can be used to treat the disease. Neutralizing antibodies proved to be a promising way to treat viral diseases, including infections with HIV, influenza virus, and other flaviviruses Thibodeaux et al., 2012;Wang et al., 2016b;Wu et al., 2015;Zhou et al., 2010). Previously, several neutralizing antibodies targeting the E protein of YFV have been isolated and show potent neutralizing activity against YFV (Daffis et al., 2005). ...
Yellow fever virus (YFV), a deadly human pathogen, is the prototype of the genus Flavivirus. Recently, YFV re-emerged in Africa and Brazil, leading to hundreds of deaths, with some cases imported to China. Prophylactic or therapeutic countermeasures are urgently needed. Previously, several human monoclonal antibodies against YFV were screened out by phage display. Here, we find that one of them, 5A, exhibits high neutralizing potency and good protection. Crystallographic analysis of the YFV envelope (E) protein in its pre- and post-fusion states shows conformations similar to those observed in other E proteins of flaviviruses. Furthermore, the structures of 5A in complex with the E protein in both states are resolved, revealing an invariant recognition site. Structural analysis and functional data suggest that 5A has high neutralization potency because it interferes with virus entry by preventing both virus attachment and fusion. These findings will be instrumental for immunogen or inhibitor design.
... To date, upon YFV infection, there are no specific and effective antiviral reagents available that can be used to treat the disease. Neutralizing antibodies proved to be a promising way to treat viral diseases, including infections with HIV, influenza virus, and other flaviviruses (Rouvinski et al., 2015;Thibodeaux et al., 2012;Wang et al., 2016b;Wu et al., 2015;Zhou et al., 2010). Previously, several neutralizing antibodies targeting the E protein of YFV have been isolated and show potent neutralizing activity against YFV (Daffis et al., 2005). ...
... Both mouse models are also suitable for antiviral screening and vaccine testing against Zika virus (ZIKV) 36 . In addition, AG129 mice were useful for testing antivirals against yellow fever virus and Japanese encephalitis virus [291][292][293] . ...
Infections with flaviviruses, such as dengue, West Nile virus and the recently re-emerging Zika virus, are an increasing and probably lasting global risk. This Review summarizes and comments on the opportunities for broad-spectrum agents that are active against multiple flaviviruses. Broad-spectrum activity is particularly desirable to prepare for the next flaviviral epidemic, which could emerge from as-yet unknown or neglected viruses. Potential molecular targets for broad-spectrum antiflaviviral compounds include viral proteins, such as the viral protease or polymerase, and host targets that are exploited by these viruses during entry and replication, including α-glucosidase and proteins involved in nucleoside biosynthesis. Numerous compounds with broad-spectrum antiviral activity have already been identified by target-specific or phenotypic assays. For other compounds, broad-spectrum activity can be anticipated because of their mode of action and molecular targets.
... Indeed, area-under-the-curve analysis showed that there was no reduction in the peak or duration of viremia, and there was no evidence that YFV-17D-specific T cells reduced disease symptoms after infection (reviewed in [34]). In contrast, passive transfer of YFV-specific immune serum provides protective immunity against lethal YFV infection in mice [28,35,36], hamsters [37], and rhesus macaques [35]. Together, these studies indicate that neutralizing antibodies are both necessary and sufficient for protection against yellow fever [33]. ...
Introduction:
The World Health Organization (WHO) and other health agencies have concluded that yellow fever booster vaccination is unnecessary since a single dose of vaccine confers lifelong immunity.
Areas covered:
We reviewed the clinical studies cited by health authorities in their investigation of both the safety profile and duration of immunity for the YFV-17D vaccine and examined the position that booster vaccination is no longer needed. We found that antiviral immunity may be lost in 1-in-3 to 1-in-5 individuals within 5 to 10 years after a single vaccination and that children may be at greater risk for primary vaccine failure. The safety profile of YFV-17D was compared to other licensed vaccines including oral polio vaccine (OPV) and the rotavirus vaccine, RotaShield, which have subsequently been withdrawn from the US and world market, respectively. Expert Commentary: Based on these results and recent epidemiological data on vaccine failures (particularly evident at >10 years after vaccination), we believe that current recommendations to no longer administer YFV-17D booster vaccination be carefully re-evaluated, and that further development of safer vaccine approaches should be considered.
... Recombinant antibodies intended for specific therapy and prevention of various viral infections have been constructed [27]. Similar antibodies are being designed for flaviviruses, such as West Nile virus [28][29][30], dengue virus type 2 [31], yellow fever virus [32], and Venezuelan equine encephalitis virus [33]. Commercially available antibodies for TBE post-exposure prophylaxis and treatment are currently absent, and development of such antibodies that would be produced in cell cultures under standard conditions is an important challenge. ...
The efficiency of several mouse monoclonal antibodies (mAbs) specific to the tick-borne encephalitis virus (TBEV) glycoprotein E in post-exposure prophylaxis was assessed, and mAb14D5 was shown to be the most active of all those studied. It was proven that the hybridoma cell line 14D5 produced one immunoglobulin H chain and two L chains. They were used to construct chimeric antibodies ch14D5a and ch14D5b, the affinity constants of which were 2.6×10(10)M(-1) and 1.0×10(7)M(-1), respectively, according to the SPR-based ProteOn biosensor assay. The neutralization index (IC50) of ch14D5a was 0.04μg/ml in the focus reduction neutralization test. In in vivo experiments, ch14D5a at a dose of 10μg/mouse resulted in a 100% survival of the mice infected with 240 LD50 of TBEV. This chimeric antibody is promising for further development of prevention and therapeutic drugs against TBEV.
... In contrast, YFV-17D-immune subjects were fully protected against YFV-17D that express the homologous envelope proteins (38,40). The protection in this case may be largely due to neutralizing antibodies since prior studies have demonstrated that adoptive transfer of immune serum alone provides partial to full protective immunity against lethal YFV in rhesus macaques (RM) (41), hamsters (42), and immunodeficient mice (43). ...
It is estimated that over 2.5 billion people are at risk for contracting dengue, a virus responsible for 50-390 million infections in addition to thousands of hospitalizations and deaths each year. There are no licensed vaccines available to combat this pathogen but substantial efforts are underway to develop live-attenuated, inactivated, and subunit vaccines that will protect against each of the four serotypes of dengue. Unfortunately, the results of a recent Phase IIb efficacy trial involving a tetravalent live-attenuated chimeric dengue virus vaccine have raised questions with regard to our current understanding of vaccine-mediated immunity to this important flavivirus. Here, we will briefly summarize these vaccination efforts and discuss the importance of informative in vivo models for determining vaccine efficacy and the need to establish a quantitative correlate of immunity in order to predict the duration of vaccine-induced antiviral protection.
... Although cellular immunity plays an important role in clearing primary WNV infection [5][6][7][8][9], memory CD8 + T cells are dispensable if high levels of antiviral antibody are present [7] and vaccine-induced memory T cells may not play a substantial role in controlling flavivirus infection in humans [10]. Moreover, a number of studies using passive immunization have shown that transfer of neutralizing antibodies to naïve animals is sufficient for protection against lethal WNV infection [11][12][13][14][15]. Accordingly, neutralizing antibody titers are generally correlated with protection against disease for licensed flavivirus vaccines. ...
West Nile virus (WNV) is a mosquito-borne flavivirus that has become endemic in the United States. From 1999-2012, there have been 37088 reported cases of WNV and 1549 deaths, resulting in a 4.2% case-fatality rate. Despite development of effective WNV vaccines for horses, there is no vaccine to prevent human WNV infection. Several vaccines have been tested in preclinical studies and to date there have been eight clinical trials, with promising results in terms of safety and induction of antiviral immunity. Although mass vaccination is unlikely to be cost effective, implementation of a targeted vaccine program may be feasible if a safe and effective vaccine can be brought to market. Further evaluation of new and advanced vaccine candidates is strongly encouraged.
... This animal model has also been shown to be valuable for initial testing of potential therapeutics to flaviviruses. We found that the humanized MAb 2C9-cIgG was able to protect AG129 mice from lethal YFV-17D challenge at a dose of ≥1.27 µg/mouse when administered 24 hours prior to infection [35]. Rodent models to study pathogenesis of JEV are available but require neonate or weanling animals in BSL-3 facilities. ...
... Appreciable results were also obtained when both murine 2C9 and 2C9-cIgG were administered 24 hours postinfection , with the survival of 70% and 20% of mice, respectively . Viral presence was not detected in surviving mice [60]. In 2005, Daffis and colleagues constructed two Abphage libraries by cloning the repertoire of YFV-infected patients. ...
More than 150 arboviruses belonging to different families are known to infect humans, causing endemic infections as well as epidemic outbreaks. Effective vaccines to limit the occurrence of some of these infections have been licensed, while for the others several new immunogens are under development mostly for their improvements concerning safety and effectiveness profiles. On the other hand, specific and effective antiviral drugs are not yet available, posing an urgent medical need in particular for emergency cases. Neutralizing monoclonal antibodies (mAbs) have been demonstrated to be effective in the treatment of several infectious diseases as well as in preliminary in vitro and in vivo models of arbovirus-related infections. Given their specific antiviral activity as well-tolerated molecules with limited side effects, mAbs could represent a new therapeutic approach for the development of an effective treatment, as well as useful tools in the study of the host-virus interplay and in the development of more effective immunogens. However, before their use as candidate therapeutics, possible hurdles (e.g., Ab-dependent enhancement of infection, occurrence of viral escape variants) must be carefully evaluated. In this review are described the main arboviruses infecting humans and candidate mAbs to be possibly used in a future passive immunotherapy.
Yellow fever virus (YFV) is an arbovirus that causes significant human morbidity and mortality. This virus has been studied intensively over the past century, although there are still no treatment options for those who become infected. Periodic and unpredictable yellow fever (YF) outbreaks in Africa and South America continue to occur and underscore the ongoing need to further understand this viral disease and to develop additional countermeasures to prevent or treat cases of illness. The use of animal models of YF is critical to accomplishing this goal. There are several animal models of YF that replicate various aspects of clinical disease and have provided insight into pathogenic mechanisms of the virus. These typically include mice, hamsters and non-human primates (NHP). The utilities and shortcomings of the available animal models of YF are discussed. Information on recent discoveries that have been made in the field of YFV research is also included as well as important future directions in further ameliorating the morbidity and mortality that occur as a result of YFV infection. It is anticipated that these model systems will help facilitate further improvements in the understanding of this virus and in furthering countermeasures to prevent or treat infections.
Diagnosis of human alphaviral infections relies on serological techniques, such as the immunoglobulin M antibody capture–enzyme-linked
immunosorbent assay (MAC-ELISA). We have humanized the alphavirus broadly cross-reactive murine monoclonal antibody 1A4B-6
to create a reagent capable of replacing human positive sera in the MAC-ELISA for diagnosis of human alphaviral infections.
Diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody
capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect IgG ELISA. Broad application of these assays is hindered
by the lack of standardized positive human control sera that react with a wide variety of flaviviruses (e.g., dengue, West
Nile, yellow fever, Japanese encephalitis, Saint Louis encephalitis, and Powassan viruses), or alphaviruses (e.g., Eastern
equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis, and chikungunya viruses) that can cause
human disease. We have created human-murine chimeric monoclonal antibodies (cMAbs) by combining the variable regions of flavivirus
(6B6C-1) or alphavirus (1A4B-6) broadly cross-reactive murine MAbs (mMAbs) with the constant region of human IgG1. These cMAbs
may be used as standardized reagents capable of replacing human infection-immune-positive control sera in indirect IgG ELISA
for diagnosis of all human flaviviral or alphaviral infections. The IgG cMAbs secreted from plasmid-transformed Sp2/0-Ag14
cells had serological activity identical to that of the parent mMAbs, as measured by ELISA using multiple flaviviruses or
alphaviruses.
Background:
Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2(c,d,e,f,g,h)) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.
Methods:
We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.
Findings:
Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.
Conclusions:
The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.
West Nile Virus (WNV) is a neurotropic flavivirus that can cause debilitating diseases, such as encephalitis, meningitis, or flaccid paralysis. We report the safety, pharmacokinetics, and immunogenicity of a recombinant humanized monoclonal antibody (MGAWN1) targeting the E protein of WNV in a phase 1 study, the first to be performed on humans. A single intravenous infusion of saline or of MGAWN1 at escalating doses (0.3, 1, 3, 10, or 30 mg/kg of body weight) was administered to 40 healthy volunteers (30 receiving MGAWN1; 10 receiving placebo). Subjects were evaluated on days 0, 1, 3, 7, 14, 21, 28, 42, 56, 91, 120, and 180 by clinical assessments, clinical laboratory studies, electrocardiograms (ECGs), and pharmacokinetic and immunogenicity assays. All 40 subjects tolerated the infusion of the study drug, and 39 subjects completed the study. One serious adverse event of schizophrenia occurred in the 0.3-mg/kg cohort. One grade 3 neutropenia occurred in the 3-mg/kg cohort. Six MGAWN1-treated subjects experienced 11 drug-related adverse events, including diarrhea (1 subject), chest discomfort (1), oral herpes (1), rhinitis (1), neutropenia (2), leukopenia (1), dizziness (1), headache (2), and somnolence (1). In the 30-mg/kg cohort, MGAWN1 had a half-life of 26.7 days and a maximum concentration in serum (C(max)) of 953 microg/ml. This study suggests that single infusions of MGAWN1 up to 30 mg/kg appear to be safe and well tolerated in healthy subjects. The C(max) of 953 microg/ml exceeds the target level in serum estimated from hamster studies by 28-fold and should provide excess WNV neutralizing activity and penetration into the brain and cerebrospinal fluid (CSF). Further evaluation of MGAWN1 for the treatment of West Nile virus infections is warranted.
Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-alpha/beta) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-alpha/beta receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6-7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-alpha/beta response may be primate-specific such that infection of mice with a functional IFN-alpha/beta antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus.
Current diagnosis of human flaviviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM)
antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Broad application of this assay is hindered by a lack of standardized
human positive-control sera that react with the wide variety of flaviviruses that can cause human disease, e.g., dengue virus
(DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus
(SLEV). We have created a human-murine chimeric antibody combining the variable regions of the broadly flavivirus cross-reactive
murine monoclonal antibody (MAb) 6B6C-1 and the constant region of human IgM to produce a standardized reagent capable of
replacing human positive-control sera in a MAC-ELISA for the diagnosis of all human flaviviral infections. The human-murine
chimeric IgM antibody secreted from plasmid-transformed Sp2/0-Ag14 cells had a level of serological activity identical to
that of 6B6C-1 as measured by ELISA, immunoblotting, and MAC-ELISA for multiple members of the flavivirus genus, including
WNV, SLEV, YFV, DENV, and JEV.
Enhancement of yellow fever virus neurovirulence for mice by specific antibody was studied with the French neurotropic vaccine strain. Experimental conditions for enhancement required mice between 14 and 40 days old and intraperitoneal administration of a selected monoclonal antibody 24 h before or up to 72 h after intracerebral virus challenge. Virus infectivity titrations were similar in brains of antibody-treated and untreated mice. Virus recovered from brains of mice with enhanced viral infections was neither qualitatively nor quantitatively different from standard virus. Humoral immune responses in enhanced infections were normal, macrophages did not become infected and viraemia was not significant. Both hydrocortisone treatment and complement depletion with cobra venom resulted in prolongation of mouse survival times but virulence enhancement persisted. Antithymocyte serum had no effect on enhancement although it reduced the humoral immune response. It is proposed that virulence enhancement is due to the combined effects of virus-specific antibody on infected cells, complement-mediated cytolysis and resultant host anti-cellular activity. There is no analogy between mechanisms effecting increased arbovirus growth in vitro in the presence of specific antibody and increased yellow fever virus neurovirulence in vivo after parenteral administration of antibody.
Several dengue (DEN) virus vaccines are in development; however, the lack of a reliable small animal model in which to test them is a major obstacle. Because evidence suggests that interferon (IFN) is involved in the human anti-DEN virus response, we tested mice deficient in their IFN functions as potential models. Intraperitoneally administered mouse-adapted DEN 2 virus was uniformly lethal in AG129 mice (which lack alpha/beta IFN and gamma IFN receptor genes), regardless of age. Immunized mice were protected from virus challenge, and survival times increased following passive transfer of anti-DEN polyclonal antibody. These results demonstrate that AG129 mice are a promising small animal model for DEN virus vaccine trials.
Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from the Alphavirus, Flavivirus, and Bunyavirus genera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.
This report describes the clinical laboratory findings in golden hamsters experimentally infected with yellow fever (YF) virus.
An accompanying paper describes the pathologic findings. Following intraperitoneal inoculation of a virulent strain of YF
virus, hamsters developed a high-titered viremia (up to 109/mL) lasting 5–6 days and abnormal liver function tests. YF hemagglutination–inhibiting
antibodies appeared 4 or 5 days after infection, often while viremia was still present. The mortality rate in YF-infected
hamsters was variable, depending on the virus strain and the age of the animals. Clinical and pathologic changes in the infected
hamsters were very similar to those described in experimentally infected macaques and in fatal human cases of YF, which indicates
that the golden hamster may be an excellent alternative animal model, in place of nonhuman primates, for research on the pathogenesis
and treatment of YF and other viscerotropic flavivirus diseases
In 1998, the Centers for Disease Control and Prevention was notified of severe illnesses and one death, temporally associated with yellow fever (YF) vaccination, in two elderly U.S. residents. Because the cases were unusual and adverse events following YF vaccination had not been studied, we estimated age-related reporting rates for systemic illness following YF vaccination. We found that the rate of reported adverse events among elderly vaccinees was higher than among vaccinees 25 to 44 years of age. We also found two additional deaths among elderly YF vaccinees. These data signal a potential problem but are not sufficient to reliably estimate incidence rates or to understand potential underlying mechanisms; therefore, enhanced surveillance is needed. YF remains an important cause of severe illness and death, and travel to disease-endemic regions is increasing. For elderly travelers, the risk for severe illness and death due to YF infection should be balanced against the risk for systemic illness due to YF vaccine.
This report updates CDC's recommendations for using yellow fever vaccine (CDC. Yellow Fever Vaccine: Recommendations of the Advisory Committee on Immunizations Practices: MMWR 1990;39[No. RR-6]1-6). The 2002 recommendations include new or updated information regarding 1) reports of yellow fever vaccine-associated viscerotropic disease (previously reported as febrile multiple organ system failure); 2) use ofyellow fever vaccine for pregnant women and persons infected with human immunodeficiency virus (HIV); and 3) concurrent use of yellow fever vaccine with other vaccines. A link to this report and other information related to yellow fever can be accessed at the website for Travelers' Health, Division of Global Migration and Quarantine, National Center for Infectious Diseases, CDC, at http://www.cdc.gov/travel/index.htm, and through the website for the Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, CDC, at http://www.cdc.gov/ncidod/ dvbid/yellowfever/index.htm.
Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.
West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.
A humanized monoclonal antibody (mAb) has been developed and its potential to protect from or cure a Venezuelan equine encephalomyelitis virus (VEEV) infection was evaluated. The VEEV-neutralizing, protective murine mAb 3B4C-4 was humanized using combinatorial antibody libraries and phage-display technology. Humanized VEEV-binding Fabs were evaluated for virus-neutralizing capacity, then selected Fabs were converted to whole immunoglobulin (Ig) G1, and stable cell lines were generated. The humanized mAb Hy4-26C, designated Hy4 IgG, had virus-neutralizing capacity similar to that of 3B4C-4. Passive antibody protection studies with purified Hy4 IgG were performed in adult Swiss Webster mice. As little as 100 ng Hy4 IgG protected 90 % of mice challenged with 100 intraperitoneal (i.p.) mean morbidity (MD(50)) doses of virulent VEEV (Trinidad donkey) 24 h after antibody transfer; also, 500 mug Hy4 IgG protected 80 % of mice inoculated with 100 intranasal MD(50) doses of VEEV. Moreover, 10 mug passive Hy4 IgG protected 70 % of mice from a VEEV challenge dose as great as 10(7) i.p. MD(50). Hy4 IgG also protected mice from challenge with another epizootic VEEV variety, 1C (P676). Importantly, therapeutic administration of the humanized mAb to mice already infected with VEEV cured 90 % of mice treated with Hy4 IgG within 1 h of VEEV inoculation and 75 % of mice treated 24 h after virus infection.
The incidence of serious and severe multisystem adverse events (AEs) following yellow fever (YF) 17D vaccine is higher in persons of advanced age. One hypothesis for the occurrence of these AEs in the elderly is immunological senescence and a reduced ability to clear the vaccine virus infection. We determined age-specific rates of serious and nonserious AEs in two large clinical trials of two YF 17D vaccines from different manufacturers. In addition, we analyzed AEs reported in a large general practice data base in the United Kingdom. Neutralizing antibody responses were compared in young and elderly subjects. In the clinical trials, involving a total of 4,532 subjects, there were no neurological and viscerotropic AEs; interestingly, the incidence of common injection site and systemic AEs was significantly lower in elderly than in younger subjects. The neutralizing antibody categorical and quantitative responses were equivalent across younger and elderly subjects. In contrast, the larger retrospective analysis of 43,555 persons receiving YF 17D in the UK general practice database revealed a higher incidence of significant neurologic and multisystem AEs with advancing age. The age-specific reporting rate ratio (RRR) was approximately twice that in the 25-44 year-old reference group for subjects in the 45-64 year age group (RRR 1.82; 95% CI 0.88,3.77) and 3-fold higher for the 65-74 year-old age group (RRR 2.82; 95% CI 0.81, 9.81). These results are consistent with previous reports on YF vaccine safety in the US (Martin M, et al. Emerg Infect Dis 2001;6:945-51; Khromova et al., Vaccine 2005;23:3256-63). In elderly persons, YF 17D vaccine is associated with a higher frequency of significant AEs in the elderly but a lower incidence of common nonserious side-effects. The neutralizing antibody response, which is the mediator of protective immunity to YF, is not diminished in healthy, elderly persons.
Current and investigational treatments of rheumatoid arthritis (RA) are described.
The current therapies used to treat RA include nonsteroidal antiinflammatory drugs (NSAIDs), used for the management of pain and inflammation; disease-modifying antirheumatic drugs (DMARDs), used as first-line therapy for all newly diagnosed cases of RA; and biological-response modifiers, targeted agents that selectively inhibit specific molecules of the immune system. Glucocorticoids and other antirheumatic drugs are also used to treat RA. DMARDs include methotrexate, hydroxychloroquine, sulfasalazine, and leflunomide. NSAIDs and glucocorticoids are effective in controlling the pain, inflammation, and stiffness related to RA. Unlike NSAIDs, they slow clinical and radiographic progression of RA. The biological-response modifiers include infliximab, etanercept, and adalimumab (inhibitors of tumor necrosis factor [TNF]-alpha); anakinra, a recombinant inhibitor of interleukin-1; abatacept, the first costimulation blocker; and rituximab, a chimeric anti-CD20 monoclonal antibody. Investigational therapies for RA include anti-interleukin-6-receptor monoclonal antibodies, new TNF-alpha inhibitors (including one for oral administration), and antibodies against proteins critical for B-cell function and survival. Data accumulated in the past decade favor early aggressive therapy for patients suspected of having RA, including early referral to a rheumatologist, new diagnostic techniques, and aggressive therapy with DMARDs, glucocorticoids, and biological agents. The benefits of this approach have been demonstrated in clinical trials.
Pharmacologic treatments of RA include NSAIDs, glucocorticoids, DMARDs, and biological agents. With an improved understanding of the pathophysiology of RA and the evidence from various clinical trials with the agents, early aggressive therapy with a combination of drugs or biological agents may be warranted for the effective treatment of RA.
This report updates CDC's recommendations for using yellow fever (YF) vaccine (CDC. Yellow fever vaccine: recommendations of the Advisory Committee on Immunizations Practices: MMWR 2002;51[No. RR-17]). Since the previous YF vaccine recommendations were published in 2002, new or additional information has become available on the epidemiology of YF, safety profile of the vaccine, and health regulations related to the vaccine. This report summarizes the current epidemiology of YF, describes immunogenicity and safety data for the YF vaccine, and provides recommendations for the use of YF vaccine among travelers and laboratory workers. YF is a vectorborne disease resulting from the transmission of yellow fever virus (YFV) to a human from the bite of an infected mosquito. It is endemic to sub-Saharan Africa and tropical South America and is estimated to cause 200,000 cases of clinical disease and 30,000 deaths annually. Infection in humans is capable of producing hemorrhagic fever and is fatal in 20%-50% of persons with severe disease. Because no treatment exists for YF disease, prevention is critical to lower disease risk and mortality. A traveler's risk for acquiring YFV is determined by multiple factors, including immunization status, location of travel, season, duration of exposure, occupational and recreational activities while traveling, and local rate of virus transmission at the time of travel. All travelers to countries in which YF is endemic should be advised of the risks for contracting the disease and available methods to prevent it, including use of personal protective measures and receipt of vaccine. Administration of YF vaccine is recommended for persons aged ≥9 months who are traveling to or living in areas of South America and Africa in which a risk exists for YFV transmission. Because serious adverse events can occur following YF vaccine administration, health-care providers should vaccinate only persons who are at risk for exposure to YFV or who require proof of vaccination for country entry. To minimize the risk for serious adverse events, health-care providers should observe the contraindications, consider the precautions to vaccination before administering vaccine, and issue a medical waiver if indicated.
Summary This report updates the 1993 recommendations by CDC's Advisory Committee on Immunization Practices (ACIP) regarding the prevention of Japanese encephalitis (JE) among travelers (CDC. Inactivated Japanese encephalitis virus vaccine: recommenda- tions of the Advisory Committee on Immunization Practices (ACIP). MMWR 1993;42(No. RR-1)). This report summarizes the epidemiology of JE, describes the two JE vaccines that are licensed in the United States, and provides recommendations for their use among travelers and laboratory workers. JE virus (JEV), a mosquito-borne flavivirus, is the most common vaccine-preventable cause of encephalitis in Asia. JE occurs throughout most of Asia and parts of the western Pacific. Among an estimated 35,000-50,000 annual cases, 20%-30% of patients die, and 30%-50% of survivors have neurologic or psychiatric sequelae. No treatment exists. For most travelers to Asia, the risk for JE is very low but varies on the basis of destination, duration, season, and activities. JE vaccine is recommended for travelers who plan to spend a month or longer in endemic areas during the JEV transmission season and for laboratory workers with a potential for exposure to infectious JEV. JE vaccine should be considered for 1) short- term (
Viral diseases transmitted by blood-feeding arthropods (arboviral diseases) are among the most important of the emerging infectious disease public health problems facing the world at the beginning of the third millennium. There are over 534 viruses listed in the arbovirus catalogue, approximately 134 of which have been shown to cause disease in humans. These are transmitted principally by mosquitoes and ticks. In the last two decades of the twentieth century, a few new arboviral diseases have been recognized. More important, however, is the dramatic resurgence and geographic spread of a number of old diseases that were once effectively controlled. Global demographic and societal changes, and modern transportation have provided the mechanisms for the viruses to break out of their natural ecology and become established in new geographic locations where susceptible arthropod vectors and hosts provide permissive conditions for them to cause major epidemics. West Nile virus is just the the latest example of this type of invasion by exotic viruses. This paper will provide an overview of the medically important arboviruses and discuss several in more detail as case studies to illustrate our tenuous position as we begin the twenty-first century.
We recently described a Venezuelan equine encephalitis virus (VEEV)-specific human monoclonal antibody (MAb), F5 nIgG, that recognizes a new neutralization epitope on the VEEV E2 envelope glycoprotein. In this study, we investigated the ability of F5 nIgG given prophylactically or therapeutically to protect mice from subcutaneous or aerosolized VEEV infection. F5 nIgG had potent ability to protect mice from infection by either route when administered 24h before exposure; however, mice treated 24h after aerosol exposure developed central nervous system infections but exhibited no clinical signs of disease. Infectious virus, viral antigen and RNA were detected in brains of both treated and untreated mice 2-6 days after aerosol exposure but were cleared from the brains of treated animals by 14-28 days after infection. This fully human MAb could be useful for prophylaxis or immediate therapy for individuals exposed to VEEV accidentally in the laboratory or during a deliberate release.
In May 2009, WHO convened a meeting of Working Group on Technical Specifications for Manufacturing and Evaluating Yellow Fever (YF) Vaccines, Geneva, Switzerland to initiate revision of the WHO Recommendations (formerly, Requirements) for YF vaccine published in WHO Technical Report Series number 872 (1998). The Working Group, consisting of experts from academia, industry, national regulatory authorities and national control laboratories, reviewed the latest issues of safety, efficacy and quality of YF vaccines and agreed that (i) the revision should focus on live attenuated YF vaccine virus 17D lineage; and that (ii) nonclinical and clinical guidelines for new vaccines prepared from 17D lineage be developed.
Yellow fever (YF) is a viral disease, endemic to tropical regions of Africa and the Americas, which principally affects humans and nonhuman primates and is transmitted via the bite of infected mosquitoes. Yellow fever virus (YFV) can cause devastating epidemics of potentially fatal, hemorrhagic disease. Despite mass vaccination campaigns to prevent and control these outbreaks, the risk of major YF epidemics, especially in densely populated, poor urban settings, both in Africa and South America, has greatly increased. Consequently, YF is considered an emerging, or reemerging disease of considerable importance. This article comprehensively reviews the history, microbiology, epidemiology, clinical presentation, diagnosis, and treatment of YFV, as well as the vaccines produced to combat YF.
Yellow fever 17D vaccine is one of the most successful vaccines ever developed and over 540 million doses have been used. Nevertheless there has been very little known about the mechanism of protection induced by the vaccine. The last couple of years have seen important advances made in understanding how the vaccine works involving studies of the innate and adaptive immune responses plus a systems biology approach. Like all vaccines, the 17D vaccine causes rare serious adverse events (SAEs) following immunization. At present, the mechanism(s) of SAEs is(are) poorly understood but our advances in understanding the immune response induced by the vaccine have promise to help elucidate the mechanism of SAEs.
: Basiliximab (B), an anti-CD25 monoclonal antibody, may represent an alternative to steroids (S) in immunosuppression after liver transplantation (LTx). The aim of this prospective randomized clinical trial was to compare B with S in a cyclosporin A (CsA)-based immunosuppression regimen in primary LTx.
: Forty-seven adult recipients of LTx were randomly assigned to receive B or S. CsA was administered at the initial dose of 10 mg/kg/day and adjusted to the target C2 level of 800 to 1000 ng/mL by day 7. Clinically suspected acute cellular rejection (ACR) was histologically confirmed. Endpoints include ACR, survival, and disease-free survival.
: In group B (26 patients), there were seven biopsy-confirmed ACR with an ACR rate of 15.4%; in group S (21 patients), 8 ACR with an ACR rate of 28.6% (P=n.s.). Cumulative survival at 36 months after transplantation was 84.3% for group B and 61.0% for group S. In hepatitis C virus patients (n=20: 12 in group B, 8 in group S), the ACR rate was 25% in group B and 50% in group S. The incidence of infection and other adverse events was similar in the two treatment groups.
: B may represent a valid alternative to S in the induction of immunosuppression in LTx. Further studies of basiliximab in a large cohort are needed.
The location of a major antigenic determinant involved in the neutralization of a flavivirus, yellow fever virus (YF), has been defined in terms of its position in the amino acid sequence of the E protein. Neutralization escape variants of the 17D vaccine strain of YF were selected with two neutralizing monoclonal antibodies. Nucleotide sequencing of the envelope protein genes (E and M) of the variants showed that in each variant there was a single nucleotide change in the E gene leading to a nonconservative amino acid substitution in the E protein at position 71 or 72. The changes are in a region of the E protein which is hydrophilic, rich in cysteine residues, and not conserved between flavivirus subgroups. Since the selecting monoclonal antibodies neutralize attenuated 17D and virulent Asibi strains of YF with equal efficiency (J. J. Schlesinger, M. W. Brandriss, and T. P. Monath, 1983, Virology 125, 8-17), it can be concluded that the neutralization determinant defined for 17D YF is also present in Asibi YF.
Antibody-dependent enhancement of yellow fever virus neurovirulence, as measured by a reduction in the average survival time of groups of mice, was demonstrated with wild-type or vaccine strains of yellow fever virus and with Japanese encephalitis virus using intraperitoneally administered monoclonal antibodies specific for the viral E glycoprotein of yellow fever virus. Enhancement of virulence could be induced by neutralizing, non-neutralizing or protective antibodies if the virus was allowed to establish a productive infection in the mouse brain before the antibody was administered. The implications of antibody-dependent enhancement in flaviviruses are discussed.
The phenomenon known as antibody-dependent enhancement (ADE) has been demonstrated in vitro but its significance in viral pathogenesis is uncertain even though it has been associated with dengue shock syndrome. Here we report for the first time the enhancement of virus virulence in mice using monoclonal antibodies (MAbs) prepared against yellow fever (YF) viruses. Our results show that the average survival time of mice was reduced by up to 33% (i.e. 6.7 to 4.5 days) and that ADE is both antibody dose-dependent and antibody- and virus strain-specific. A total of 12 YF viruses and 11 MAbs were examined and of these only three YF viruses (FNV, Asibi and B11) could be enhanced in vivo by only two MAbs (427 and 126). A particular combination of virus and antibody is required for ADE to take place.
Immunization of monkeys with yellow fever virus-specified nonstructural protein NS1 resulted in protection against fatal hepatitis as well as marked reduction in the magnitude of viremia after subcutaneous challenge with yellow fever virus. The results may be relevant to the design of possible subunit or recombinant flavivirus vaccines.
Monoclonal antibodies to the envelope proteins (E) of the 17D vaccine strain of yellow fever virus (17D YF) and to dengue 2 virus were examined for their ability to confer passive protection against lethal 17D YF encephalitis in mice. All 13 IgG anti-17D YF antibodies, regardless of neutralizing capacity, conferred solid protection when given in a relatively high dose prior to intracerebral inoculation of virus. Three antibodies with high in vitro neutralizing titres were all protective at a low dose as were several non-neutralizing antibodies. One flavivirus group-reactive antibody to dengue 2 virus conferred similar protection at low dose. Protection was also observed when antibodies were given several days after virus inoculation when peak infectious virus titres and histopathological evidence of infection were present in brains. The ability of a non-neutralizing antibody to protect could not be attributed to complement-dependent lysis of virus-infected cells and did not correlate with avidity or with proximity of its binding site to a critical neutralizing epitope of the E protein. Some antibodies, characterized as non-neutralizing by plaque reduction assay on Vero cells, inhibited the growth of virus in a mouse neuroblastoma cell line, suggesting one possible mechanism of protection. These results may be relevant to the design of prospective flavivirus vaccines and support the possibility of conferring broadened protection among flaviviruses by stimulating the antibody response to appropriate epitopes of the E protein.
Nineteen monoclonal antibodies were produced to the 17D strain of yellow fever virus (17D YF). Virus-specific structural and nonstructural proteins were identified for 17D YF and for the parent wild Asibi YF by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fourteen of the monoclonal antibodies were directed against the envelope glycoprotein, E, and five against the nonstructural protein gp 48. The E protein of 17D YF was resolved as a double complex whereas the E protein of Asibi YF appeared as a single band of slightly lower molecular weight. The only IgM anti-E antibody obtained precipitated and neutralized 17D YF specifically with no activity against Asibi YF. This antibody also distinguished clearly by neutralization (N) between the 17D-204 derived vaccine strain to which the animal had been immunized and 17D YF strains of different origin. All 13 IgG anti-E monoclonal antibodies had hemagglutination-inhibition (HI) activity to 17D YF and all but one neutralized Asibi YF; however, only 3 of the 13 neutralized 17D YF. Four anti-E antibodies cross-reacted with other flaviviruses by HI or HI and N. Three of the five anti-gp 48 antibodies had complement-fixation (CF) titers against 17D YF and Asibi YF but none had N or HI activity.
We have identified and characterized eight antigenic epitopes on the 53,000 dalton envelope (E) glycoprotein of Saint Louis encephalitis (SLE) virus by using monoclonal antibodies. One of these epitopes (E-1c) encoded for the type-specific biologic functions of hemagglutination (HA) and neutralization (N). Injection of 50 ng of anti-E-1c antibody protected the majority of mice from peripheral challenge with 100 i.p. LD50 of SLE virus. Similar levels of protection with antibodies specific for other epitopes usually required greater than or equal to 1000-fold additional antibody. Attempts to block N or protection at the E-1c antigenic domain by using antibody to several other SLE epitopes that strongly competed for the E-1c site were unsuccessful. Enhancement of protection was observed with mixtures of the more cross-reactive antibodies. The E-1c antibody was also effective in abrogating SLE virus replication until neural invasion occurred. On the basis of these findings, the topologic arrangement and function of the eight SLE E glycoprotein epitopes on the virion spike is proposed.
Sixteen monoclonal antibodies that reacted with the envelope glycoprotein (E) of 17D vaccine strain yellow fever virus (17D YF), including two antibodies produced against dengue 2 virus, were used in a solid phase competitive binding assay (CBA) to define spatial relationships among antigenic determinants on 17D YF E. The antibodies showed YF strain, type or flavivirus group specificities and nine epitopes were identified on 17D YF E by patterns of neutralization, haemagglutination inhibition and competition of antibody binding. Epitopes defined by neutralizing antibodies with strain and type specificities appeared spatially distant but competition between type-specific neutralizing antibodies and some non-neutralizing antibodies against type and group determinants suggested close proximity among epitopes in these regions. Despite competition between some neutralizing and non-neutralizing monoclonal antibodies in CBA, plaque assays revealed no interference with neutralization by non-neutralizing antibody.
Eleven different hybridoma cell lines that produce monoclonal antibodies directed against the three Sindbis virus structural proteins, (capsid, E1, and E2) have been isolated by fusing spleen cells from Sindbis virus immunized BALB/c mice with the mouse myeloma cell line SP2/0-Ag14. The initial screening for antibody-producer hybridomas was carried out with an enzyme-linked immunosorbant assay (ELISA) that could detect as little as 10 ng antigen and 2.5 μg hybridoma antibody. The antibodies were characterized further by radioimmune precipitation with [35S]methionine-labeled viral antigens, by SDS-polyacrylamide gel electrophoresis, and by isoelectric focusing of [35S]methionine-labeled hybridoma antibodies. Antibody class and subclass was determined by solid phase radioimmune assay. Of the 11 clones analyzed, 2 produced anti-El IgG, 1 made an anti-E2 IgG, 3 produced anti-capsid IgG, and 3 formed anti-capsid Ighl antibodies. One clone made an IgG that reacted with intact virus but not with any of the isolated proteins, and one clone produced an Ighl that reacted with all three virus proteins as well as bovine serum albumin.
The 17D vaccine strain of yellow fever virus (17D-YF) produces a safe human arboviral infection that can provide antisera of well-defined specificity under chronologically defined conditions. We studied 17D-YF growth in human peripheral blood macrophages and in two continuous Fc receptor-bearing, macrophage-like cell lines, P388D1 of mouse origin and U937 of human origin. Cells were infected with virus in the presence or absence of antibody to 17D-YF and to two related flaviviruses, St. Louis encephalitis (SLE) and dengue 2 (D2V). The virus 17D-YF grew in the three cell types when infection was established without antibody; viral yields were increased by addition of antibody to 17D-YF, SLE, and D2V. Increased titers of virus were accompanied by an increased number of infected cells by immunofluorescent assay. Enhancing activity was present in the IgG but not the IgM fractions of immune sera. Infection without cytopathic effect was observed in U937.
Monoclonal antibodies (MAbs) prepared against the yellow fever virus (YF) vaccine strain 17D (17D YF) envelope E protein were used to investigate Fc piece involvement in antibody-mediated protection against YF encephalitis in mice 17D YF passaged either in Vero cells or in mouse brain (P-YF) to increase neurovirulence was used. To avoid uncertainty concerning antibody clearance and blood-brain barrier penetration, and to directly compare protective activity with neutralization in vitro, pre-formed antibody-virus complexes were injected intracerebrally or assayed for plaque formation in parallel. F(ab')2 fragments of an IgG2a MAb that strongly neutralized both YF strains retained molar equivalent neutralizing activity in vitro, but did not protect. However, further incubation of such F(ab')2-virus antibody complexes with rabbit IgG, but not F(ab')2 anti-mouse IgG resulted in protection. To unambiguously test for Fc piece involvement in this model we derived an IgG2a isotype switch variant from a protective IgG1 MAb-secreting hybridoma and prepared F(ab')2 fragments of the derivative. Intact and fragmented antibodies exhibited weak neutralizing activity. The variant antibody failed to protect against P-YF, but against considerably less neurovirulent 17D YF its protective capacity was 10-fold higher than that of its IgG1 parent. F(ab')2 fragments of the variant did not protect. Together, these results provide strong evidence of an in vivo protective function for the anti-virion antibody Fc piece and indicate that in vitro neutralizing activity as a predictor of antibody protective capacity is dependent on Fc piece integrity and isotype.
In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.
Rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA) is the first monoclonal antibody approved by the US Food and Drug Administration for the treatment of cancer. It is a genetically engineered chimeric (murine-human) monoclonal antibody (mAb) directed against the CD20 antigen found on the surface of normal and malignant B cells. Multicenter studies have demonstrated its efficacy against relapsed low-grade and follicular non-Hodgkin's lymphoma (NHL). The mAb demonstrated tolerable side effects, primarily limited to fevers and chills associated with the first infusion. The currently recommended dosage is 375 mg/m2/infusion, given weekly for 4 weeks. Because of its human component, rituximab has low immunogenicity and should not significantly hinder future retreatment. Future studies will evaluate the antitumor activity of rituximab combined with various other chemotherapeutic or biologic agents in the treatment of B-cell lymphoma and other CD20-positive lymphoid neoplasms.
The Kabat Database was initially started in 1970 to determine the combining site of antibodies based on the available amino
acid sequences at that time. Bence Jones proteins, mostly from human, were aligned, using the now-known Kabat numbering system,
and a quantitative measure, variability, was calculated for every position. Three peaks, at positions 24–34, 50–56 and 89–97,
were identified and proposed to form the complementarity determining regions (CDR) of light chains. Subsequently, antibody
heavy chain amino acid sequences were also aligned using a different numbering system, since the locations of their CDRs (31–35B,
50–65 and 95–102) are different from those of the light chains. CDRL1 starts right after the first invariant Cys 23 of light
chains, while CDRH1 is eight amino acid residues away from the first invariant Cys 22 of heavy chains. During the past 30
years, the Kabat database has grown to include nucleotide sequences, sequences of T cell receptors for antigens (TCR), major
histocompatibility complex (MHC) class I and II molecules and other proteins of immunological interest. It has been used extensively
by immunologists to derive useful structural and functional information from the primary sequences of these proteins. An overall
view of the Kabat Database and its various applications are summarized here. The Kabat Database is freely available at http://immuno.bme.nwu.edu
Subadult and adult hamsters were inoculated intraperitoneally with 106 TCID50 of yellow fever (YF) virus (Jimenez strain). Four animals from each group were subjected daily to histologic examination
for 9 days. The liver showed spotty necrosis on day 3 after infection, which was followed by steatosis and focally confluent
necrosis. In surviving hamsters, hepatocyte regeneration began on day 8, which was accompanied by decreasing steatosis. The
spleen initially exhibited lymphoid hyperplasia, which was followed by lymphoid depletion and increased phagocytosis by splenic
macrophages. Focal pancreatic acinar necrosis and spotty adrenal cortical necrosis were seen transiently between days 5 and
7. Viral antigen was detected immunohistochemically in the liver and the spleen. TUNEL analysis showed a dynamic change of
hepatocyte necrapoptosis, with activity corresponding to the severity of disease. The histopathologic changes were more severe
in younger (subadult) animals. The YF-hamster model appears to be an accurate and inexpensive experimental system for studying
the pathophysiology and treatment of YF
The outbreak of West Nile (WN) encephalitis in the United States has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. Although equine WN vaccines are currently being developed, a WN vaccine for humans is years away. There is also no specific therapeutic agent for flaviviral infections. The incidence of human WN virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the vaccine's economic feasibility. It has been shown, however, that prophylactic application of antiflaviviral antibody can protect mice from subsequent virus challenge. This model of antibody prophylaxis using murine monoclonal antibodies (MAbs) has been used to determine the timing of antibody application and specificity of applied antibody necessary for successful prophylaxis. The major flaviviral antigen is the envelope (E) glycoprotein that binds cellular receptors, mediates cell membrane fusion, and contains an array of epitopes that elicit virus-neutralizing and nonneutralizing antibodies. The protective efficacy of an E-glycoprotein-specific MAb is directly related to its ability to neutralize virus infectivity. The window for successful application of prophylactic antibody to prevent flaviviral encephalitis closes at about 4 to 6 days postinfection concomitant with viral invasion of the brain. Using murine MAbs to modify human disease results in a human antimouse antibody (HAMA) response that eventually limits the effectiveness of subsequent murine antibody applications. To reduce the HAMA response and make these MAbs more generally useful for humans, murine MAbs can be "humanized" or human MAbs with analogous reactivities can be developed. Antiflaviviral human or humanized MAbs might be practical and cost-effective reagents for preventing or modifying flaviviral diseases.
During the past 20 years there has been a dramatic resurgence or emergence of epidemic arboviral diseases affecting both humans and domestic animals. These epidemics have been caused primarily by viruses thought to be under control such as dengue, Japanese encephalitis, yellow fever, and Venezuelan equine encephalitis, or viruses that have expanded their geographic distribution such as West Nile and Rift Valley fever. Several of these viruses are presented as case studies to illustrate the changing epidemiology. The factors responsible for the dramatic resurgence of arboviral diseases in the waning years of the 20th century are discussed, as is the need for rebuilding the public health infrastructure to deal with epidemic vector-borne diseases in the 21st century.
Ribavirin was evaluated as a potential treatment of yellow fever (YF) in a hamster model of the disease. Ribavirin treatment during the first five days after YF virus infection improved survival, reduced tissue damage in target organs (liver and spleen), prevented hepatocellular steatosis, and normalized alanine aminotransferase levels. The results of this study suggest that ribavirin may be effective in the early treatment of YF, and that its mechanism of action in reducing liver pathology in YF virus infection may be similar to that observed with ribavirin in the treatment of chronic hepatitis C virus infection.
Since 1996, the scientific community has become aware of 14 reports of yellow fever vaccine (YEL)-associated viscerotropic disease (YEL-AVD) cases and four reports of YEL-associated neurotropic disease (YEL-AND) worldwide, changing our understanding of the risks of the vaccine. Based on 722 adverse event reports after YEL submitted to the U.S. Vaccine Adverse Event Reporting System in 1990-2002, we updated the estimates of the age-adjusted reporting rates of serious adverse events, YEL-AVD and YEL-AND. We found that the reporting rates of serious adverse events were significantly higher among vaccinees aged > or =60 years than among those 19-29 years of age (reporting rate ratio = 5.9, 95% CI 1.6-22.2). Yellow fever is a serious and potentially fatal disease. For elderly travelers, the risk for severe illness and death due to yellow fever infection should be balanced against the risk of a serious adverse event due to YEL.
In recent years, a growing number of serious adverse events (including deaths) associated with the yellow fever (YF) vaccine has been reported. If YF vaccination were incorporated in routine programs, administered to children, the risk of deaths from this vaccine would be minimized provided that mortality of children vaccinated below 1 year were negligible. However, in affected areas the vaccine is administered to all age groups. This poses a dilemma to public health authorities - what proportion of a population subject to low risk of YF outbreaks should be vaccinated in order to minimize the total number of serious adverse events (including deaths) due both to natural infection and vaccination? In other words, how much vaccination is safe? Our results suggest that, depending on the age-specific rates of developing vaccine-induced serious adverse events and the risk of yellow fever outbreaks, the optimum proportion to vaccinate may be lower than the proportion that would prevent an epidemics or even be zero. We also show that the vaccine should not be applied to individuals older than 60 years of age because the risk of serious adverse events (including deaths) is higher for that age class. Our work is instrumental to the discussion on the optimum strategy to vaccinate affected populations against yellow fever. Therefore, the aim of this work is to estimate the optimum proportion to vaccinate against YF taking into account the risks of serious adverse events associated with both the vaccine and natural infection.
Although the live attenuated yellow fever (YF) 17D vaccine is considered to be one of the safest vaccines in the world today, several cases of disease associated with administration of the vaccine have been reported, including YF vaccine-associated viscerotropic disease (YF-VAVD), which was first described in 1996. All YF-VAVD isolates sequenced to date have shown very little genomic change when compared to their parental vaccine strains. In this study, we report the characterization of an isolate, BeH291597 (Brazil75), from a 1975 fatal case of YF-VAVD in Brazil. Comparison of Brazil75 with the genomic sequence of the parental 17DD vaccine strain revealed two amino acid substitutions (at positions M-49 and NS4B-240) that were unique to Brazil75. Although still a rare occurrence, this isolate suggests that YF-VAVD has been present much longer than previously recognized.
Yellow fever vaccine-associated viscerotropic disease (YEL-AVD) is a recently described severe adverse event after yellow fever vaccination, and some cases have been reported in different countries [Anonymous. Effects of yellow fever and vaccination. Lancet 2001;358(9296):1907-9]. Herein we describe a YEL-AVD case in a young woman, who died after vaccination with 17D-204 strain. Clinical, serological and immunochemical analysis as well as virus detection, quantification, sequence analysis and cytokine release, were performed. Further investigations on yellow fever vaccine adverse events, and carefully analysis of the immune response elicited are important tasks for the future.
Using a recently described hamster model of yellow fever (YF), we compared the hematologic and clinical chemistry changes that occur in blood with the histopathologic alternations observed in liver and other organs. Inflammatory foci and necroapoptotic hepatocytes were first observed in the liver three days after YF infection. This was accompanied by a rapid increase in serum transaminase and bilirubin values, elevation of prothrombin times, thrombocytopenia, and leukocytosis. Maximum liver pathology was observed on the sixth and seventh days post-infection; this corresponded to the peak alternations in clinical chemistry and hematologic values. In surviving hamsters, regenerating hepatocytes began to appear on the eighth day post-infection; this was accompanied by a corresponding return to baseline levels of most of the aforementioned clinical laboratory values. The histopathologic and clinical laboratory findings in the hamster model were very similar to those observed in severe human cases of YF. These results provide further validation of the utility of the hamster model for studying the pathogenesis and treatment of YF.
Humans infected with West Nile virus (WNV) may clinically present with symptoms that are suggestive of neurological infection. Nearly all treatments of WNV disease have been effective in animal models only if administered before or soon after viral challenge. Here, we evaluated whether a potent neutralizing anti-WNV humanized monoclonal antibody (MAb), hE16, could improve the course of disease in a hamster model when administered after the virus had infected neurons in the brain. Five days after viral injection, WNV was detected in the brains of hamsters by cytopathic assay, quantitative reverse-transcription polymerase chain reaction, and immunohistochemical staining of WNV envelope in neurons. Notably, 80%-90% of the hamsters treated 5 days after viral injection by intraperitoneal injection with hE16 survived the disease, compared with 37% of the placebo-treated hamsters (P< or =.001). The hamsters that received hE16 directly in the brain also exhibited markedly improved survival rates, compared with those in the placebo-treated hamsters. In prospective experiments, hamsters with high levels of infectious WNV in their cerebrospinal fluid were also protected by hE16 when administered 5 days after viral injection. These experiments suggest that humanized MAbs with potent neutralizing activity are a possible treatment for human patients after WNV has infected neurons in the central nervous system.
Antiviral compounds were evaluated for efficacy against yellow fever virus (YFV) in a hamster model of YFV-induced liver disease. Challenge with a 10(2) 50% cell culture infectious doses of YFV resulted in a 50-80% mortality rate in female hamsters. Virus was detected by quantitative real-time RT-PCR (QRT-PCR) in liver, kidney, spleen and serum with peak titers on 4-6 days post-viral challenge (dpi). Serum levels of alkaline phosphatase, alanine aminotransferase (ALT), bilirubin, blood urea nitrogen, potassium and creatinine were significantly elevated, while serum levels of albumin, amylase, glucose, calcium, globulin, phosphorus, sodium and total protein were significantly reduced. Packed cell volume and white blood cell count were significantly elevated during the course of the infection. Intraperitoneal treatment of hamsters with 0.5-5 microg/kg/day interferon (IFN) alfacon-1, 100mg/kg/day viramidine or 50 mg/kg/day ribavirin, initiated 4h prior to YFV challenge, resulted in significant improvement in survival and serum ALT levels. Treatment with IFN alfacon-1 or ribavirin starting 2dpi, also significantly improved survival and serum ALT levels in hamsters challenged with YFV. Pre- and post-virus exposure treatment with IFN alfacon-1 was efficacious in improving disease in YFV-infected hamsters.