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Third-generation smallpox vaccines: Challenges in the absence of clinical smallpox
Abstract
Smallpox, a disease caused by variola virus, is estimated to have killed hundreds of millions to billions of people before it was certified as eradicated in 1980. However, there has been renewed interest in smallpox vaccine development due in part to zoonotic poxvirus infections and the possibility of a re-emergence of smallpox, as well as the fact that first-generation smallpox vaccines are associated with relatively rare, but severe, adverse reactions in some vaccinees. An understanding of the immune mechanisms of vaccine protection and the use of suitable animal models for vaccine efficacy assessment are paramount to the development of safer and effective smallpox vaccines. This article focuses on studies aimed at understanding the immune responses elicited by vaccinia virus and the various animal models that can be used to evaluate smallpox vaccine efficacy. Harnessing this information is necessary to assess the effectiveness and potential usefulness of new-generation smallpox vaccines.
... Designing a novel type of vaccine is vital since monkeypox has spread to multiple continents, from Africa to Europe and the Americas. As such, the vaccine should be available for prophylactic and therapeutic (post-exposure) use [115,116]. ...
... The viral strains were propagated without genetic manipulations on cell culture under controlled conditions and were purified using the plaque-purification method. Vaccines such as ACAM2000 (derived from Dryvax) were prepared on Vero cells, and for Elstree-BN (derived from Lister/Elstree), chick embryo fibroblast cells were used [115][116][117]. Since the second-generation vaccine viral strains show close relatedness to the strains used for first-generation vaccines, they can induce a similar reactogenicity to the live, animalderived vaccines. ...
... The murine model was used to evaluate the immunobiology of VACV, ECTV, CPXV, and MPXV. Independent of the murine model, the impact of MPXV on the host was assessed using monkeys, dormice, ground squirrels, and black-tailed prairie dogs as animal models [115,[124][125][126][127][128][129][130][131][132]. ...
The eradication of smallpox was an enormous achievement due to the global vaccination program launched by World Health Organization. The cessation of the vaccination program led to steadily declining herd immunity against smallpox, causing a health emergency of global concern. The smallpox vaccines induced strong, humoral, and cell-mediated immune responses, protecting for decades after immunization, not only against smallpox but also against other zoonotic orthopoxviruses that now represent a significant threat to public health. Here we review the major aspects regarding orthopoxviruses’ zoonotic infections, factors responsible for viral transmissions, as well as the emerging problem of the increased number of monkeypox cases recently reported. The development of prophylactic measures against poxvirus infections, especially the current threat caused by the monkeypox virus, requires a profound understanding of poxvirus immunobiology. The utilization of animal and cell line models has provided good insight into host antiviral defenses as well as orthopoxvirus evasion mechanisms. To survive within a host, orthopoxviruses encode a large number of proteins that subvert inflammatory and immune pathways. The circumvention of viral evasion strategies and the enhancement of major host defenses are key in designing novel, safer vaccines, and should become the targets of antiviral therapies in treating poxvirus infections.
... This cell-derived Lister vaccine showed no severe complications in clinical trials in Asia and Europe [14]. Other VACV vaccines that were generated using Lister as the parental strain, such as the Elstree-BN, were passaged in chicken embryo cells and manufactured for public use by Bavarian Nordic after preclinical studies in non-human primates yielded a safe and immunogenic profile [56][57][58]. Sanofi Pasteur developed the Lister/CEP (chicken embryo primary cell) vaccine by passaging the live animal-derived Lister vaccine three times in chicken embryo primary cells, which proved to have comparable safety and immunogenicity to the parental vaccine strain [59]. VACV vaccines that originate from the NYCBH strain had a rough start after the first candidate (grown in cell culture) failed to demonstrate an adequate take rate in 1968. ...
... Despite the early failure, this strain was further passaged in MRC-5 cells and used to generate the Cell Culture Smallpox Vaccine [14]. A different vaccine, ACAM2000®which is currently licensed in the USA was derived from Dryvax (an NYCBH strain) grown in Vero cells [57]. Another descendent of the NYCHB strain, WR, is a neurovirulent strain that has been widely passaged in rabbits, mice, and cell culture [14]. ...
... Cell-and tissue-derived smallpox vaccines can induce reactogenicity similar to that found in live animal-derived vaccines. Even though many of these vaccines have not been directly confronted by smallpox, they will likely retain the same efficacy as the live animal-derived vaccines due to their close relation [57]. ...
Various vaccinia virus (VACV) strains were applied during the smallpox vaccination campaign to eradicate the variola virus worldwide. After the eradication of smallpox, VACV gained popularity as a viral vector thanks to increasing innovations in genetic engineering and vaccine technology. Some VACV strains have been extensively used to develop vaccine candidates against various diseases. Modified vaccinia virus Ankara (MVA) is a VACV vaccine strain that offers several advantages for the development of recombinant vaccine candidates. In addition to various host-restriction genes, MVA lacks several immunomodulatory genes of which some have proven to be quite efficient in skewing the immune response in an unfavorable way to control infection in the host. Studies to manipulate these genes aim to optimize the immunogenicity and safety of MVA-based viral vector vaccine candidates. Here we summarize the history and further work with VACV as a vaccine and present in detail the genetic manipulations within the MVA genome to improve its immunogenicity and safety as a viral vector vaccine.
... A unique feature of smallpox vaccine research is that safer vaccines are sought by assessing strain immunogenicity in the absence of an endemic outbreak [16][17][18] 1970s [16][17][18]. Therefore, a vaccine was evaluated by comparing the immunological responses of the target vaccine with firstgeneration vaccine(s) and by assessing the protection conferred in animal challenge models [16][17][18]. ...
... A unique feature of smallpox vaccine research is that safer vaccines are sought by assessing strain immunogenicity in the absence of an endemic outbreak [16][17][18] 1970s [16][17][18]. Therefore, a vaccine was evaluated by comparing the immunological responses of the target vaccine with firstgeneration vaccine(s) and by assessing the protection conferred in animal challenge models [16][17][18]. ...
... A unique feature of smallpox vaccine research is that safer vaccines are sought by assessing strain immunogenicity in the absence of an endemic outbreak [16][17][18] 1970s [16][17][18]. Therefore, a vaccine was evaluated by comparing the immunological responses of the target vaccine with firstgeneration vaccine(s) and by assessing the protection conferred in animal challenge models [16][17][18]. Three clinical studies, including one post-marketing surveillance study of LC16m8, have been performed in the United States and Japan since 2002. ...
... Well-known examples are the eradicated smallpox and the ongoing mpox epidemic. Smallpox had been with humans for centuries [27,28], killing hundreds of millions of people before being declared eradicated by the World Health Or ganization (WHO) in the late 20th century [29][30][31]. Unfortunately, mpox resurfaced jus as COVID-19 was in decline, and the virus was declared a public health emergency o international concern by the WHO in 2022 [32][33][34], suggesting that a new poxvirus epi demic may be underway. ...
... Well-known examples are the eradicated smallpox and the ongoing mpox epidemic. Smallpox had been with humans for centuries [27,28], killing hundreds of millions of people before being declared eradicated by the World Health Organization (WHO) in the late 20th century [29][30][31]. Unfortunately, mpox resurfaced just as COVID-19 was in decline, and the virus was declared a public health emergency of international concern by the WHO in 2022 [32][33][34], suggesting that a new poxvirus epidemic may be underway. ...
Poxviruses have been associated with humans for centuries. From smallpox to mpox to lumpy skin disease virus (LSDV), members of the poxvirus family have continued to threaten the lives of humans and domestic animals. A complete understanding of poxvirus-mediated cellular processes will aid in the response to challenges from the viruses. In this study, we demonstrate that LSDV infection results in an abnormal ultrastructure of the endoplasmic reticulum (ER) lumen in primary bovine embryonic fibroblast (BEF) cells, and we further show that an ER imbalance occurs in LSDV-infected BEF cells. Additionally, we believe that ER stress-related apoptosis plays a role in the late apoptosis of BEF cells infected with LSDV, primarily through the activation of the CCAAT/enhancer binding protein homologous protein (CHOP)-Caspase-12 signal. In addition to cell apoptosis, a further investigation showed that LSDV could also activate autophagy in BEF cells, providing additional insight into the exact causes of LSDV-induced BEF cell death. Our findings suggest that LSDV-induced BEF cell apoptosis and autophagy may provide new avenues for laboratory diagnosis of lumpy skin disease progression and exploration of BEF cell processes.
... The advantage of the rabbitpox model is the ability to produce a natural aerosol transmission of the virus between animals with secondary lesions, although the rabbitpox infection does not occur in humans. Other models comprising Orthopoxvirus in the literature include VV, ectromelia, cowpox, and monkeypox virus in mice [22]. Interestingly, besides VV, monkeypox and cowpox [23] also infect humans. ...
... Interestingly, besides VV, monkeypox and cowpox [23] also infect humans. Although mice models can be used in studies that are practical and can reach statistical power, the differences in immune system responses between mice and humans can result in differences in epitope recognition, thus hampering translatability to humans [22]. Notably, the Ind-3a strain of VARV was explored by the Institute of Cancer Research (ICR) in SCID mice [24]. ...
The Committee for Orphan Medicinal Products (COMP) of the European Medicines Agency (EMA) is responsible for evaluating applications for orphan designation (OMPDs) and for deciding the orphan status at the time of marketing authorization of medicines in the European Union (EU). In this review, we provide transparency regarding assessment criteria and an in-depth review of nonclinical models and data that have been used to support OMPDs. Additionally, we present a literature-based analysis of existing nonclinical models and discuss key features of nonclinical studies, which are considered crucial for the support of future OMPDs. This could not only inform future drug development in rare infectious conditions, but also indicate areas where nonclinical models are indispensable or can be made more efficient.
... The Japanese have developed a third generation vaccine named LC16m8, derived from first generation Lister strain vaccinia. While there are fewer published trials than with MVA, they have used LC16m8 extensively, and have experienced few serious complications (39,40,41,42). This vaccine would be good for outbreak control. ...
... Research on development of third and fourth generation vaccines will probably progress. Vaccinia and its many artificial variants such as MVA and NYVAC are large stable DNA viruses, relatively safe and easy to work with (4, 41,42). Given their safety in humans, they may be excellent vectors for other vaccine antigens. ...
The potential use of smallpox as a weapon for bioterrorism has created a need for more and better smallpox vaccines. The first generation vaccines such as Dryvax were produced by crude methods that would not allow licensure today. Second generation vaccines, grown in modern safe tissue cultures but employing seed virus from first generation vaccines of known effectiveness, have been developed. One, ACAM2000, has been licensed and added to the US National Stockpile. These second generation vaccines can produce the same complications as first generation vaccines, and myopericarditis has been well documented as associated with ACAM2000. This has created advocacy for third and fourth generation smallpox vaccines. Third generation vaccines are those which have been attenuated either by serial passage in non-human cells, or by careful deletions of genes in the laboratory. Two of these, MVA (Modified Vaccinia Ankara), and LC16m8 (a derivative of Lister strain vaccinia), have been tested in various human trials. These seem to be ready to apply for licensure if there proves to be a market. Fourth generation vaccines, created as subunits of full strength vaccinia, or fully engineered non-replicating molecules which express various known epitopes of vaccinia and/or smallpox, have also been developed. It may be difficult or impossible to prove the efficacy of these vaccines because smallpox no longer exists and there is no animal model that accurately reflects the human disease. These fourth generation vaccines include several large stable DNA viruses into which immunogens from others agents such as HIV and malaria can be inserted, so they may have a future as vector vaccines for a variety of other agents besides smallpox.
... First-generation vaccines used 40 years ago do not meet current safety, potency and tolerance standards required by regulatory bodies. Therefore, second and third generation vaccines have been developed over the years, but to a limited extent because the main purpose has been to increase the vaccine stockpile of smallpox biodefense programs in some countries [42,43]. In this context, the monkeypox outbreak in 2022 surprised the world as the number of cases increased exponentially worldwide and exposed the urgent need to expand our vaccine options against orthopoxvirus infection [25]. ...
The Wyeth strain of vaccinia virus (VACV) produced by Wyeth Pharmaceuticals was supposedly used to manufacture the old freeze-dried American smallpox vaccine, Dryvax, until its discontinuation in 2008. Although the genomic sequences of numerous Dryvax clones have been reported, data on VACV-Wyeth genomes are still lacking. Genomic analysis of old VACV strains is relevant to understand the evolutionary relationships of smallpox vaccines, particularly with the recent resumption of smallpox vaccination in certain population groups as an attempt to control the worldwide monkeypox outbreak. Here we analyzed the complete genome sequences of three VACV-Wyeth clonal isolates obtained from a single seed vial donated to the Brazilian eradication program in the 1970s. Wyeth clones show >99.3% similarity to each other and >95.3% similarity with Dryvax clones, mapping together in clade I of the vaccinia group. Although the patterns of SNPs and INDELs comparing Dryvax and Wyeth clones are overall uniform, important differences were detected particularly at the ends of the genome. In addition, we detected recombinant events of clone Wyeth A111 and the Dryvax clone Acam2000, suggesting that other regions of the genomes may have similar patchy patterns of recombination. A small-scale serological survey using VACV-Wyeth as antigen in ELISA assays revealed that 63 of the 65 individuals born before the end of smallpox vaccination in Brazil still have anti-VACV IgG antibodies, demonstrating the usefulness of the VACV-Wyeth strain in future extended serological studies of the Brazilian population.
... Multiple studies in animal models have shown that LC16m8 protects the host from viral challenges, demonstrating the drug's safety and efficacy. [44][45][46][47][48] Smallpox immunization with an injectable vaccine called modified vaccinia Ankara (MVA) is approved for use in the European Union (marketed as IMVANEX) and Canada (marketed as IMVAMUNE). The FDA has authorized the use of modified live attenuated vaccine (MVA; brand name: JYNNEOSTM) in 2019 for the prevention of both smallpox and MPX in populations at high risk of infection. ...
Monkeypox (MPX) is a zoonotic disease that is endemic to the western and central regions of Africa and it is caused by monekypox virus (MPXV), which is classified as a member of the Poxviridae family, specifically the Chordopoxvirinae subfamily, and the Orthopoxvirus genus. The current multiregional outbreak of MPX, which started in May of 2022, has since swiftly spread across the globe and thus has been declared a global public health emergency by the World Health Organization (WHO). Protective immunity against MPXV can be achieved by administering a smallpox vaccination, as the two viruses share antigenic properties. Although smallpox was declared eradicated in 1980, the vaccine campaign was halted the following year, leaving the population with significantly less immunity than it had before. The potential for human-to-human transmission of MPXV has grown as a result. Due to the lack of a particular treatment for MPX infection, anti-viral medications initially designed for the smallpox virus are being employed. However, the prognosis for MPX may vary depending on factors like immunization history, pre-existing illnesses, and comorbidities, even though the majority of persons who develop MPX have a mild, self-limiting illness. Vaccines and antiviral drugs are being researched as potential responses to the latest 2022 MPX epidemic. The first-generation smallpox vaccinations maintained in national stockpiles of several countries are not recommended due to not meeting the current safety and manufacturing criteria, as stated by the WHO. Newer, safer (second- and third-generation) smallpox vaccines, such as JYNNEOSTM, which has been licensed for the prevention of MPX, are indicated as potentially useful in the interim guideline. Studies on vaccines and antiviral drugs are still being investigated as possible remedies to the recent MPX outbreak. This mini-review article serves as a retrospective look at the evolution of smallpox vaccines from their inception in the 1700s to the current trends upto the end of year 2022 specifically for developing monkeypox vaccines.
... The current smallpox vaccine has several contraindications that include up to 30% of the US population, making widespread use of this vaccine problematic. Safer, attenuated and subunit smallpox vaccines (third-generation smallpox vaccines) [102,103] are in various stages of research and development. A deeper understanding of the role that cytokines play in immune responses to poxviruses will allow us to more readily find the delicate balance between immunogenicity and reactogenicity. ...
Smallpox was eradicated 34 years ago due to the success of the smallpox vaccine; yet, the vaccine continues to be studied because of its importance in responding to potential biological warfare and the adverse events associated with current smallpox vaccines. Interindividual variations in vaccine response are observed and are, in part, due to genetic variation. In some cases, these varying responses lead to adverse events, which occur at a relatively high rate for the smallpox vaccine compared with other vaccines. Here, we aim to summarize the cytokine responses associated with smallpox vaccine response to date. Along with a description of each of these cytokines, we describe the genetic and adverse event data associated with cytokine responses to smallpox vaccination.
... The safety profile of candidate new smallpox vaccines can be established in the clinic, but in the absence of clinical smallpox, evaluation of efficacy of new-generation smallpox vaccines poses a challenge. Efficacy evaluation will rely heavily on data obtained from appropriate animal models, as well as bridging of preclinical immunogenicity and efficacy data to immunogenicity data obtained in clinical studies [12]. Complicating efficacy evaluation, particularly for smallpox vaccines that are fundamentally different from vaccines used for eradication of smallpox, is that the correlates of protective immunity to smallpox are not known [13]. ...
Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.
The eradication of smallpox is considered one of the greatest public health achievements of the twentieth century. Specimens of smallpox virus are, however, still officially held in the United States and Russia.
Samples of variola DNA may also be recoverable from old medical samples, such as the century-old smallpox scabs discovered in an envelope tucked in a 19th century medical textbook in a New Mexico library in 2004. In 2014, U.S. official found more smallpox samples in a storage room on the National Institutes for Health campus in Bethesda, Maryland.
The World Health Organization (WHO) declared smallpox eradicated in 1980. The last confirmed naturally occurring smallpox case was in 1977/ Ali Maow Maalin, a hospital employee in Merca, Somalia, survived his bout with smallpox.
Following eradication, the World Health Organization requested that all laboratories in the world either destroy their smallpox virus stocks or transfer them to one of two reference laboratories, the Institute of Viral Preparations in Moscow or the United States Centers for Disease Control and Prevention in Atlanta, Georgia. The stocks of the Institute of Viral Preparations were transferred in 1994 to the State Research Center of Virology and Biotechnology of the Russian Federation in Siberia, now the WHO Collaborating Centre for Orthopoxvirus Diagnostics. (download to read more)
Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.
Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates.
We conducted a clinical trial of the safety and immunogenicity of modified vaccinia Ankara (MVA) to examine the effects of dose and route of administration.
Seventy-two healthy, vaccinia virus-naive subjects received 1 of 6 regimens of MVA (ACAM3000) or placebo consisting of 2 administrations given 1 month apart.
MVA was generally well tolerated at all dose levels and by all routes. More pronounced local reactogenicity was seen with the intradermal and subcutaneous routes than with intramuscular administration. Binding antibodies to whole virus and neutralizing antibodies to the intracellular mature virion and extracellular enveloped virion forms of vaccinia virus were elicited by all routes of MVA administration and were greater for the higher dose by each route. Similar levels of neutralizing antibodies were seen at a 10-fold-lower dose given intradermally (1 x 10(7) median tissue culture infective doses [TCID(50)]), compared with responses after 1 x 10(8) TCID(50) given intramuscularly or subcutaneously. T cell immune responses to vaccinia virus were detected by an interferon gamma enzyme-linked immunospot assay but had no clear relationship to dose or route.
These data suggest that intradermal immunization with MVA provides a dose-sparing effect by eliciting antibody responses similar in magnitude and kinetics to those elicited by the intramuscular or subcutaneous routes but at a 10-fold-lower dose.
Despite the success of smallpox vaccination, the immunological correlates of protection are not fully understood. To investigate this question, we examined the effect of immunization with modified vaccinia Ankara (MVA) on subsequent challenge with replication-competent vaccinia virus (Dryvax).
Dryvax challenge by scarification was conducted in 36 healthy subjects who had received MVA (n = 29) or placebo (n = 7) in a previous study of doses and routes of immunization. Subjects were followed up for clinical take, viral shedding, and immune responses.
MVA administration attenuated clinical takes in 21 (72%) of 29 subjects, compared with 0 of 7 placebo recipients (P = .001). Attenuation was most significant in MVA groups that received 1 x 10(7) median tissue culture infective doses (TCID(50)) intradermally (P = .001) and 1 x 10(7) TCID(50) intramuscularly (P = .001). Both duration and peak titer of viral shedding were reduced in MVA recipients. Peak neutralizing antibody responses to vaccinia virus or MVA previously induced by MVA immunization were associated with attenuated takes (P = .02) and reduced duration (P = .001) and titer (P = .005) of viral shedding.
MVA immunization results in clinical and virologic protection against Dryvax challenge. Protection is associated with prior induction of neutralizing antibodies to MVA or vaccinia virus. MVA administered intradermally has protective and immunologic responses similar to those of a 10-fold-higher dose given subcutaneously.
Vaccinia virus (VACV) was used as the vaccine strain to eradicate smallpox. VACV is still administered to healthcare workers or researchers who are at risk of contracting the virus, and to military personnel. Thus, VACV represents a weapon against outbreaks, both natural (e.g., monkeypox) or man-made (bioterror). This virus is also used as a vector for experimental vaccine development (cancer/infectious disease). As a prototypic poxvirus, VACV is a model system for studying host-pathogen interactions. Until recently, little was known about the targets of host immune responses, which was likely owing to VACVs large genome (>200 open reading frames). However, the last few years have witnessed an explosion of data, and VACV has quickly become a useful model to study adaptive immune responses. This review summarizes and highlights key findings based on identification of VACV antigens targeted by the immune system (CD4, CD8 and antibodies) and the complex interplay between responses.
Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola, the etiological agent of smallpox. In humans, MPXV
causes a disease similar to smallpox and is considered to be an emerging infectious disease. Moreover, the use of MPXV for
bioterroristic/biowarfare activities is of significant concern. Available small animal models of human monkeypox have been
restricted to mammals with poorly defined biologies that also have limited reagent availability. We have established a murine
MPXV model utilizing the STAT1-deficient C57BL/6 mouse. Here we report that a relatively low-dose intranasal (IN) infection
induces 100% mortality in the stat1−/− model by day 10 postinfection with high infectious titers in the livers, spleens, and lungs of moribund animals. Vaccination
with modified vaccinia virus Ankara (MVA) followed by a booster vaccination is sufficient to protect against an intranasal
MPXV challenge and induces an immune response more robust than that of a single vaccination. Furthermore, antiviral treatment
with CMX001 (HDP-cidofovir) and ST-246 protects when administered as a regimen initiated on the day of infection. Thus, the
stat1−/− model provides a lethal murine platform for evaluating therapeutics and for investigating the immunological and pathological
responses to MPXV infection.
Smallpox was a deadly disease when it was rife yet despite its eradication more than 30 years ago, the possibility of accidental or intentional release has driven research in search of better definitions of correlates of protective immunity. Mousepox, a disease caused by ectromelia virus (ECTV), is arguably one of the best surrogate small animal models for smallpox. Correlates of protection in mousepox are well defined during primary infection, whereas those in a secondary infection, which have definite relevance to vaccination strategies, are less well understood. We previously established that neutralizing antibody (Ab), which is generated far more rapidly during a secondary infection compared with a primary infection, has a key role during a secondary virus challenge. In this study, we show that the route of immunization or the use of homologous or heterologous virus vaccines for immunization does not influence the ability of mice to control high-dose virulent ECTV challenge or to mount a substantial secondary neutralizing Ab response. In contrast, the recall cytotoxic T lymphocyte (CTL) responses generated under these regimes of immunization were varied and did not correlate with virus control. Furthermore, unlike the recall Ab response that was generated rapidly, the kinetics of the secondary antiviral CTL response was no different to a primary infection and peaked only at day 8 post-challenge. This finding further underscores the importance of Ab in conferring protection during secondary poxvirus infection. This information could potentially prove useful in the design of safer and more efficacious vaccines against poxviruses or other diseases using poxvirus vectors.
Characterization of field isolates of viruses associated with pox-like outbreaks involving both cows (cattle) and buffaloes was carried out. PCR and electron microcopy of representative virus isolates from these animals, initially identified them as orthopoxviruses (OPXVs). Sequence and phylogenetic analyses of A-type inclusion and haemagglutinin (HA) genes of these isolates revealed a closer relationship with other OPXVs. Sequencing of the HA gene of these isolates revealed sequence identity of 96.2-99.8 and 94.6-98.7% at the nucleotide and deduced amino acid level, respectively, with VACVs, particularly with VACV-MVA, a vaccine strain. Further, C18L (ankyrin repeat protein)-gene-based BPXV-specific PCR confirmed them as BPXV. It is apparent from this study that pox-like outbreaks in cows and buffaloes in India are caused, in most cases, by BPXV. Considering the zoonotic implications of buffalopox, such outbreaks involving both buffaloes and cows in a mixed flock may pose a significant public health threat. Transmissibility of BPXV between different species including cows, buffaloes and human beings implies the potential reemergence of the virus in the subcontinent, similar to vaccinia-like outbreaks witnessed recently in other countries.
Previous studies of CD8(+) T cell immunodominance after primary virus infection of F(1) mice compared with their inbred parents have generally concluded that no dramatic changes occur. In this study, we revisit this issue using vaccinia virus (VACV), which has a large genome, a recently defined immunodominance hierarchy in mice, and is a candidate vector for vaccines. We found that immunogenicity of VACV peptides defined using inbred mice was highly variable in F(1) progeny: some peptides were equally immunogenic in F(1) and inbred, whereas others elicited responses that were reduced by >90% in F(1) mice. Furthermore, the dominance of a peptide in the relevant inbred parent did not predict whether it would be poorly immunogenic in F(1) mice. This result held using F(1) hybrids of MHC-congenic mice, suggesting that MHC differences alone were responsible. It was also extended to foreign epitopes expressed by an rVACV vaccine. F(1) mice were less able to mount responses to the poorly immunogenic peptides when used as a sole immunogen, ruling out immunodomination. In addition, conserved TCR Vbeta usage between inbred and F(1) mice did not always correlate with strong responses in F(1) mice. However, direct estimation of naive precursor numbers showed that these were reduced in F(1) compared with inbred mice for specificities that were poorly immunogenic in the hybrids. These data have implications for our understanding of the extent to which MHC diversity alters the range of epitopes that are immunogenic in outbred populations.
Smallpox has been eradicated but stockpiles of the causative infectious agent, variola virus, have been maintained over decades. Today, the threat of accidental or intentional poxvirus release is accompanied by the fact that the existing licensed smallpox vaccines cause rare but severe adverse reactions yet are the only products with approved efficacy against smallpox. New safer vaccines and new strategies of immunization are to be developed to fit to a scenario of emergency smallpox vaccination. However, we still lack knowledge about the pathogen and the mechanisms involved in acquiring protective immunity. Here, we review the history of smallpox vaccines and recent achievements in the development of highly efficacious and safer vaccines and vaccine applications. These include i) assessment of adequate animal models to study pathogenesis and protective immunity, ii) characterization of the immunity elicited by next generation vaccines, and (iii) the investigation of the requirements for rapidly protective vaccination.
1. A dermal strain of vaccine virus has been adapted to a simple culture medium consisting of minced chick embryo suspended in Tyrode's solution. 2. The bacteria-free culture virus, thus obtained, produces in lower animals and in man typical vaccinia that renders them refractory to infection with ordinary vaccine virus harvested from calves.
As yet, the only human infectious disease eradicated from our planet is smallpox, caused by variola virus a member of the poxvirus family. The vaccination success, with the declaration by WHO in 1980 of a worldwide free of smallpox, was largely due to the availability of a quite effective and stable live vaccine, as well as the restricted human host for virus infection. Variola was considered one of the most devastating diseases of human mankind. With the sudden appearance of the HIV/AIDS in 1981, an infection which spread rapidly to become a pandemic in a short time, causing up to date more than 22 million deaths, about 40 million people infected and a current incidence of about 3 million deaths per year, this dreadful pandemic has become one of the most severe diseases in the World, specially in poor countries. While different antiviral drugs have been developed that block virus replication at various stages of infection, however the rapid virus escape that follows during the drug therapy due to mutations, makes the development of vaccines the most secure option to control and eradicate the disease. Numerous vaccines have been developed, but to date the clinical trials have failed to show any efficacy against HIV infection. Due to the proven success of vaccinia virus in the control of smallpox as well as of poxvirus recombinants against veterinary diseases, a major effort has been directed to document the advantages of poxvirus vectors as vaccines against multiple diseases. Two of the most promising poxvirus vectors are the highly attenuated modified vaccinia virus Ankara (MVA) and the modified Copenhagen strain NYVAC. In this commentary I describe the biological characteristics of the attenuated poxvirus vectors, MVA and NYVAC, with emphasis in their application in HIV preclinical and clinical trials, and considerations as future HIV vaccines.
Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host's immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity approximately 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.
The immune response elicited by LC16m8, a candidate smallpox vaccine that was developed in Japan by cold selection during serial passage of the Lister vaccine virus in primary rabbit kidney cells, was compared to Dryvax in a mouse model. LC16m8 carries a mutation resulting in the truncation of the B5 protein, an important neutralizing target of the extracellular envelope form of vaccinia virus (EV). LC16m8 elicited a broad-spectrum immunoglobulin G (IgG) response that neutralized both EV and the intracellular mature form of vaccinia virus and provoked cell-mediated immune responses, including the activation of CD4+ and CD8+ cells, similarly to Dryvax. Mice inoculated with LC16m8 had detectable but low levels of anti-B5 IgG compared to Dryvax, but both Dryvax and LC16m8 sera neutralized vaccinia virus EV in vitro. A truncated B5 protein (approximately 8 kDa) was expressed abundantly in LC16m8-infected cells, and both murine immune sera and human vaccinia virus immunoglobulin recognized the truncated recombinant B5 protein in antigen-specific enzyme-linked immunosorbent assays. At a high-dose intranasal challenge (100 or 250 50% lethal doses), LC16m8 and Dryvax conferred similar levels of protection against vaccinia virus strain WR postvaccination. Taken together, the results extend our current understanding of the protective immune responses elicited by LC16m8 and indicate that the relative efficacy in a mouse model rivals that of previously licensed smallpox vaccines.
The presence of zoonotic poxviruses in nature represents a potential human health risk that has to be re-evaluated by health authorities not only in developing countries, but also in many developed countries. For example, buffalopox virus infection remains to be a threat to humans and cattle in India, and monkeypox virus infection persists in several inhabited places in Africa and, more recently, in the USA. There are also a great number of zoonotic transmissions of cowpox virus from cats to humans in Europe. For almost a decade in Brazil, vaccinia-like viruses have been isolated from human and cattle infections. This review examines the ability of potentially pathogenic orthopoxviruses, including feral versions of vaccinia virus vaccine, to persist in nature and re-emerge for reasons we do not yet understand.
The attenuated, tissue-cultured, third-generation smallpox vaccine LC16m8 was administered to vaccinia-naive infants in Japan during the 1970s without serious adverse events. It is a good candidate for use as part of a prevention plan for bioterrorism.
To assess the immunogenicity and frequency of adverse events of LC16m8 vaccine in unvaccinated and previously vaccinated adults.
Between 2002 and 2005 we vaccinated and revaccinated 1529 and 1692 adults, respectively, in the Japan Self-Defense Forces with LC16m8 vaccine, given intraepidermally using a bifurcated needle. Vaccinees were examined 10 to 14 days after vaccination to determine if they had developed a major skin reaction ("take"). Neutralizing antibody responses among 200 participants were assessed using a plaque-reduction neutralization test 30 days postvaccination. We monitored vaccinees for adverse events for 30 days postvaccination.
Documentation of a vaccine take, presence of neutralizing antibody response, and frequency of adverse events.
The proportions of take in vaccinia-naive and previously vaccinated individuals were 1443 of 1529 (94.4% [95% confidence interval {CI}, 93.2%-95.9%] and 1465 of 1692 (86.6% [95% CI, 85.0%-88.2%]), respectively. Seroconversion or an effective booster response among the individuals with take was elicited in 37 of 41 (90.2% [95% CI, 81.2%-99.3%]) vaccinia-naive participants and in 93 of 155 (60.0% [95% CI, 52.3%-67.7%]) previously vaccinated participants. One case of allergic dermatitis and another of erythema multiforme, both of which were mild and self-limited, were suspected to be caused by vaccination. No severe adverse events were observed.
Administration of an attenuated tissue-cultured smallpox vaccine (LC16m8) to healthy adults was associated with high levels of vaccine take and seroconversion in those who were vaccinia-naive and yielded an effective booster response in some previously vaccinated individuals.
Bovine vaccinia is an exanthematic disease caused by Vaccinia virus (VACV). This zoonosis has been associated with several cases of bovine infection, particularly in milk herds. Farmers, milkers and their close contacts developed lesions on the hands, forearms, legs and face accompanied by fever, headache, malaise, myalgia and axillary, inguinal and cervical lymphadenopathy. VACV infections have a significant public health impact due to their occupational character, high frequency of transmission and the improper medical treatment often applied.
To study natural human infection by VACV and to analyze clinical and epidemiological aspects, emphasizing the patients' immunological status.
Ninety-eight individuals from rural properties with bovine vaccinia (BV) outbreaks who were at risk due to contact were submitted to epidemiological and clinical studies. From these individuals, 54 sera were analyzed by serological and molecular procedures. This study was conducted in Rio de Janeiro State from September 2002 to October 2006.
The clinical frequency of infection was 52.0%, with 57.4% ELISA and 43.0% PRNT-positive reactions. DNAemia was detected in 18.5% of the analyzed sera, and 50% of smallpox-vaccinated individuals developed symptoms.
This study confirms the high clinical frequency of human VACV infection, even among vaccinated individuals. The infection was related to detection of IgG- or IgM-specific antibodies that correlates in most of the cases with positive PRNT. The DNAemia suggests viremia during VACV natural infections. Our data indicate that patients vaccinated against smallpox may no longer be protected.
Multiple monkeypox virus (MPXV) animal models have been discussed in previous studies, but no small animal models, nor most non-human primate models, demonstrated the protracted asymptomatic incubation phase seen in systemic human orthopoxvirus illness. Herein, we characterize a black-tailed prairie dog (PD) (Cynomys ludovicianus) model of infection, via intranasal and intradermal exposures, with the two MPXV clades. Daily observations of the animals were made (food consumption, general symptoms, disease presentation), while weights and virus evaluations (ocular, nasal, oropharyngeal, faeces, blood) were obtained/made every third day. Generalized rash became apparent 9-12 days post-infection for all animals. Individual animals demonstrated a range of symptoms consistent with human monkeypox disease. Measurable viraemias and excretas were similar for both clade-representative strains and persisted until at least day 21. Greater morbidity was observed in Congo Basin strain-challenged animals and mortality was observed only in the Congo Basin strain-challenged animals. The PD model is valuable for the study of strain-dependent differences in MPXV. Additionally, the model closely mimics human systemic orthopoxvirus disease and may serve as a valuable non-human surrogate for investigations of antivirals and next generation orthopoxvirus vaccines.
We report the second case of severe postvaccinial encephalitis with acute disseminated encephalomyelitis since smallpox vaccination
was reintroduced in 2002. Both affected patients responded dramatically with early intervention of intravenous immunoglobulin.
Our patient, who also received concurrent vaccinia immunoglobulin and corticosteroids, demonstrated full recovery.
Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4(+) lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.
The antibody responses elicited by immunization of humans with vaccinia virus (VACV) strains Lister, Dryvax and NYVAC have been determined and compared. Neutralizing antibodies against intracellular mature virus (IMV) and extracellular enveloped virus (EEV), and binding antibody titres (ELISA) against the EEV protein B5, the IMV proteins A27 and H3, and VACV-infected cell lysate were measured. Lister and Dryvax induced broadly similar antibody titres, consistent with the fact that these vaccines each protected against smallpox. In contrast, antibody titres induced by NYVAC were significantly lower than those induced by both Lister and Dryvax. Moreover, there were qualitative differences with NYVAC-immunized subjects failing to induce A27-specific antibodies. These observations suggest that although NYVAC is a safer VACV strain, it does not induce an optimal VACV-specific antibody response. However, NYVAC strains engineered to express antigens from other pathogens remain promising candidate vaccines for immunization against other diseases.
The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.
Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed.
The MVA virus is a lab virus ideally suited for vaccination of both man and animal which can be differentiated from the known Vaccinia strains by the use of numerous biological markers. Its reduced virulence for the chick embryo, for experimental animals and for man is a particularly characteristic feature. With the exception of chick embryo fibroblasts, the MVA virus grows in cell cultures only abortively. This applies particularly to cells of human origin in which the cytopathic effect and plaque formation are completely missing. The restriction analysis of the DNS of the MVA virus demonstrates that its genetic structure differs from that of the CVA basic virus and other orthopox viruses. In contrast to the WHO reference strain Elstree, the MVA virus has a genome shortened by about 9%. The use of the MVA virus for human vaccination is particularly indicated in persons to be vaccinated for the first time and likely to entail a risk (on account of allergies etc.) because it brings about a state of revaccination without complications. The MVA virus can be administered in intracutaneous, subcutaneous or intramuscular injections. Innocuousness and successful vaccination have been demonstrated in more than 120,000 persons. While other Vaccinia strains, such as the Elstree virus, experience a drastic increase of virulence in the immunosuppressed organism (subjected to whole-body irradiation), the MVA virus cannot be activated not even in this situation.
Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed.
Vaccination played a leading role in transforming the social and political status of medicine in Japanese society in the second half of the nineteenth century. The process began well before the Meiji Restoration of 1868 created a centralized government under the Japanese emperor. At the beginning of the century, medicine was a private business. There was no oversight from an interested government, and there were no medical societies or journals in which to debate and formulate opinion about medical practice. Medical knowledge was transmitted privately through personal lineage structures whose members jealously guarded their medical techniques. For almost a half century before live vaccine could be imported, knowledge of vaccination was limited to a small group of Japanese physicians who could read Dutch. This special knowledge created a medical elite whose members managed the transmission of vaccination after the vaccine arrived, and dominated the new medical and public health bureaucracies created by the Meiji state. By the end of the century, a rigorous vaccination program was in place, smallpox mortality had fallen, and Japan's Western-oriented physicians were in control of a national public health bureaucracy that could monitor the vaccination status of individuals in households throughout Japan.
Smallpox produced the death of up to thirty percent of those infected, so Jenner's preventive method spread quickly. The Spanish government designed and supported a ten-year effort to carry smallpox vaccine to its American and Asian territories in a chain of arm-to-arm vaccination of children. An expedition directed by Doctor Francisco Xavier de Balmis sailed from Corunna in November 1803, stopping in the Canary Islands, Puerto Rico, and Venezuela. Balmis led a subexpedition to Cuba, Mexico, and the Philippines; his assistants returned to Mexico in 1807, while Balmis took vaccine to China and returned to Spain (and again to Mexico, 1810-13). Vice-director José Salvany and his staff took vaccine to present-day Colombia, Ecuador, Peru, Bolivia, and Chilean Patagonia. The Spanish Royal Philanthropic Vaccine Expedition shows the first attempts to solve questions still important for the introduction of new immunizations--professionalization in public health, technology transfer, protection of research subjects, and evaluation of vaccine efficacy, safety, and cost.
Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third- and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence.
In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.
SUMMARY Extracellular rabbitpox virus released naturally from infected cells differed antigenically from intracellular virus released by artificial disruption of cells. Intracellular virus was neutralized by antiserum prepared against live rabbitpox virus and by antiserum against inactivated vaccinia virus. In contrast, extracellular virus was neutralized only by rabbitpox antiserum. The antibodies responsible for the neutralization of intracellular and extracellular virus could be absorbed separately from rabbitpox antiserum. Morphologically, extracellular virus differed from intracellular virus in possessing an outer envelope. This envelope was probably the site of the virus antigen characteristic of extracellular virus, and fluorescent antibody staining of infected cells suggested that it was derived from the modified host cell membrane. Antibody directed against extracellular virus was responsible for the ability of rabbitpox antiserum to control the spread of rabbitpox virus in tissue culture and probably for its ability to protect rabbits from rabbitpox infection. Extracellular virus should therefore be used as the test virus in titrations of neutral- izing antibody if these are to assess the protective activity of an antiserum.
To combat variola virus in bioterrorist attacks, it is desirable to develop a noninvasive vaccine. Based on the vaccinia Tiantan (VTT) strain, which was historically used to eradicate the smallpox in China, we generated a modified VTT (MVTT(ZCI)) by removing the hemagglutinin gene and an 11,944bp genomic region from HindIII fragment C2L to F3L. MVTT(ZCI) was characterized for its host cell range in vitro and preclinical safety and efficacy profiles in mice. Despite replication-competency in some cell lines, unlike VTT, MVTT(ZCI) did not cause death after intracranial injection or body weight loss after intranasal inoculation. MVTT(ZCI) did not replicate in mouse brain and was safe in immunodeficient mice. MVTT(ZCI) induced neutralizing antibodies via the intranasal route of immunization. One time intranasal immunization protected animals from the challenge of the pathogenic vaccinia WR strain. This study established proof-of-concept that the attenuated replicating MVTT(ZCI) may serve as a safe noninvasive smallpox vaccine candidate.
Oral rabies vaccination of target reservoir species has proved to be one of the pillars of successful rabies elimination programs. The use of live attenuated rabies virus vaccines has been extensive but several limitations hamper its future use. A recombinant vaccinia-rabies vaccine has also been successfully used for the oral vaccination of several species. Nevertheless, its lack of efficacy in certain important rabies reservoirs and concerns on the use of this potent live virus as vaccine carrier (vector) impair the expansion of its use for new target species and new areas. Several attenuated and host-restricted poxvirus alternatives, which supposedly offer enhanced safety, have been investigated. Once again, efficacy in certain target species and innocuity through the oral route remain major limitations of these vaccines. Alternative recombinant vaccines using adenovirus as an antigen delivery vector have been extensively investigated and may provide an important addition to the currently available oral rabies vaccine repertoire, but are not the primary subject of this review.
Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 x 10(6)pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.
Infection of rabbits with aerosolized rabbitpox virus (RPXV) produces a disease similar to monkeypox and smallpox in humans and provides a valuable, informative model system to test medical countermeasures against orthopoxviruses. Due to the eradication of smallpox, the evaluation of the efficacy of new-generation smallpox vaccines depends on relevant well-developed animal studies for vaccine licensure. In this study, we tested the efficacy of IMVAMUNE [modified vaccinia Ankara-Bavarian Nordic (MVA-BN)] for protecting rabbits against aerosolized RPXV. Rabbits were vaccinated with either phosphate-buffered saline (PBS), Dryvax, a single low dose of IMVAMUNE, a single high dose of IMVAMUNE, or twice with a high dose of IMVAMUNE. Aerosol challenge with a lethal dose of RPXV was performed 4 weeks after the last vaccination. All PBS control animals succumbed to the disease or were euthanized because of the disease within 7 days postexposure. The rabbits vaccinated with Dryvax, a low dose of IMVAMUNE, or a single high dose of IMVAMUNE showed minimal to moderate clinical signs of the disease, but all survived the challenge. The only clinical sign displayed by rabbits that had been vaccinated twice with a high dose of IMVAMUNE was mild transient anorexia in just two out of eight rabbits. This study shows that IMVAMUNE can be a very effective vaccine against aerosolized RPXV.
Vaccinia virus (VACV) induces a vigorous virus-specific CD8+ T cell response that plays an important role in control of poxvirus infection. To identify immunodominant poxvirus proteins and to facilitate future testing of smallpox vaccines in non-human primates, we used an algorithm for the prediction of VACV peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 VACV ORFs; 100 of these peptides did not contain the canonical proline at position three of the Mamu-A*01 binding motif. Cellular immune responses in PBMC from two vaccinia-vaccinated Mamu-A*01+ macaques were assessed by IFNgamma ELISPOT assays. Vaccinated macaques recognized 17 peptides from 16 different ORFs with 6 peptides recognized by both macaques. Comparison with other orthopoxvirus sequences revealed that 12 of these epitopes are strictly conserved between VACV, variola, and monkeypoxvirus. ELISPOT responses were also observed to eight epitopes that did not contain the canonical P3 proline. These results suggest that the virus-specific CD8+ T cell response is broadly directed against multiple VACV proteins and that a subset of these T cell epitopes is highly conserved among orthopoxviruses.
In spite of the eradication of smallpox over 30 years ago; orthopox viruses such as smallpox and monkeypox remain serious public health threats both through the possibility of bioterrorism and the intentional release of smallpox and through natural outbreaks of emerging infectious diseases such as monkeypox. The eradication effort was largely made possible by the availability of an effective vaccine based on the immunologically cross-protective vaccinia virus. Although the concept of vaccination dates back to the late 1800s with Edward Jenner, it is only in the past decade that modern immunologic tools have been applied toward deciphering poxvirus immunity. Smallpox vaccines containing vaccinia virus elicit strong humoral and cellular immune responses that confer cross-protective immunity against variola virus for decades after immunization. Recent studies have focused on: establishing the longevity of poxvirus-specific immunity, defining key immune epitopes targeted by T and B cells, developing subunit-based vaccines, and developing genotypic and phenotypic immune response profiles that predict either vaccine response or adverse events following immunization.
In 2002, the US Federal government initiated a campaign to vaccinate military personnel and members of the civilian population against smallpox to counter a possible bioterrorism attack. More than 1,200,000 military personnel and approximately 40,000 civilians have been vaccinated since that time. The incidence of myopericarditis in these vaccinees has clearly exceeded calculated background rates and has prompted discussion about cardiac inflammation and other potential vaccine-associated cardiac complications such as dilated cardiomyopathy (DCM) and myocardial ischemia. Although it seems reasonable to predict that some cases of smallpox-associated myopericarditis will progress to DCM, only small numbers have been documented so far, and a causal relationship is difficult to ascertain. With regard to myocardial ischemia, both historical and current data do not suggest a causal association with the vaccine. We describe a case report of myopericarditis following smallpox immunization and provide a review of all cardiac complications associated with vaccination.
The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.
Subunit vaccine candidates against poxvirus infection induced protective humoral and cellular response in animal models but their immunogenicity in human remains unknown. We have therefore evaluated in vitro the CD4 T cell response of the major antigens B5R and A33R and characterized their CD4 T cell epitopes. Twelve peptides selected on the basis of their binding capacity to HLA-DR molecules, induced CD4 T lymphocytes harvested in healthy donors. In the A33R proteins two peptides are T cell stimulating for at least half of the donors and are restricted to multiple HLA-DR molecules in agreement with their broad specificity for HLA-DR molecules. In B5R, two peptides exhibited a good immunoprevalence but only one is a good binder to multiple HLA-DR molecules. One peptide was a moderate binder for multiple HLA-DR molecules, although it was efficiently presented to peptide-specific T cell lines. Altogether, our data demonstrated the capacity of B5R and A33R peptides to elicit a T cell response in multiple healthy donors and showed that promiscuity and immunoprevalence of CD4 T cell epitopes are not necessarily associated.
Currently, more than half of the world's population has no immunity against smallpox variola major virus. This phase I double-blind, randomized trial was conducted to compare the safety and immunogenicity of two clonally derived, cell-culture manufactured vaccinia strains, ACAM1000 and ACAM2000, to the parent vaccine, Dryvax. Thirty vaccinia-naïve subjects were enrolled into each of three groups and vaccines were administered percutaneously using a bifurcated needle at a dose of 1.0x10(8)PFU/mL. All subjects had a primary skin reaction indicating a successful vaccination. The adverse events, 4-fold neutralizing antibody rise and T cell immune responses were similar between the groups.
Intratracheal (i.t.) infection of mice with cowpox virus (CPXV), is lethal at a lower dose than intranasal (i.n.) inoculation. CPXV deleted for cytokine response modifier A (CPXVDeltacrmA) was attenuated compared to CPXV after i.t. inoculation. This attenuation could not be attributed to differences in virus replication, immunomodulators, or cells infiltrating the lungs. Deletion of crmA also caused attenuation during intradermal (i.d.) infection. In contrast to i.t.-inoculated virus, deletion of crmA reduced virus replication at the site of infection. This difference correlated to increased numbers of CD3(+) cells in CPXVDeltacrmA-associated dermal lesions. Thus, crmA is a virulence factor in mice during either pulmonary or dermal cowpox infection; however the influence of crmA is more evident during i.d. inoculation. This suggests that the host immune response differs in the two routes of infection and emphasizes the need to consider the effect of route of infection when examining functions of virulence factors in vivo.
Suitable animal models are needed to study monkeypox virus (MPXV) as human monkeypox clinically resembles smallpox and MPXV is a zoonotic and potential bioterroristic agent. We have demonstrated that a species of African dormice, Graphiurus kelleni, is susceptible to a lethal infection of MPXV and that MPXV replicated in multiple organs of this species. Following intranasal administration, MPXV replicated locally in the nasal mucosa causing necrosis and hemorrhage with subsequent systemic spread to lymph nodes, spleen, liver, and other tissues where it caused severe necrosis and/or hemorrhage leading to death. The dormouse model was validated for testing prophylactic (Dryvax vaccine) and therapeutic (cidofovir) test articles against intranasal challenges with MPXV.
The prevention of infectious disease via prophylactic immunization is a mainstay of global public health efforts. Vaccine design would be facilitated by a better understanding of the type and durability of immune responses generated by different vaccine vectors. We report here the results of a comparative immunogenicity trial of six different vaccine vectors expressing the same insert antigen, cowpox virus B5 (CPXV-B5). Of those vectors tested, recombinant adenovirus (rAd5) was the most immunogenic, inducing the highest titer anti-B5 antibodies and conferring protection from sublethal vaccinia virus challenge in mice after a single immunization. We tested select heterologous prime-boost combinations and identified recombinant vesicular stomatitis virus (rVSV) and recombinant Venezuelan equine encephalitis virus replicons (VRP) as the most synergistic regimen. Comparative data such as those presented here are critical to efforts to generate protective vaccines for emerging infectious diseases as well as for biothreat agents.
Vaccinia virus is the smallpox vaccine. It is the most intensively studied poxvirus, and its study has provided important insights about virus replication in general and the interactions of viruses with the host cell and immune system. Here, the entry, morphogenesis and dissemination of vaccinia virus are considered. These processes are complicated by the existence of two infectious vaccinia virus particles, called intracellular mature virus (IMV) and extracellular enveloped virus (EEV). The IMV particle is surrounded by one membrane, and the EEV particle comprises an IMV particle enclosed within a second lipid membrane containing several viral antigens. Consequently, these virions have different biological properties and play different roles in the virus life cycle.
The success of the World Health Organization smallpox eradication program three decades ago resulted in termination of routine vaccination and consequent decline in population immunity. Despite concerns regarding the reintroduction of smallpox, there is little enthusiasm for large-scale redeployment of licensed live vaccinia virus vaccines because of medical contraindications and anticipated serious side effects. Therefore, highly attenuated strains such as modified vaccinia virus Ankara (MVA) are under evaluation in humans and animal models. Previous studies showed that priming and boosting with MVA provided protection for >2 years in a monkeypox virus challenge model. If variola virus were used as a biological weapon, however, the ability of a vaccine to quickly induce immunity would be essential. Here, we demonstrate more rapid immune responses after a single vaccination with MVA compared to the licensed Dryvax vaccine. To determine the kinetics of protection of the two vaccines, macaques were challenged intravenously with monkeypox virus at 4, 6, 10, and 30 days after immunization. At 6 or more days after vaccination with MVA or Dryvax, the monkeys were clinically protected (except for 1 of 16 animals vaccinated with MVA), although viral loads and number of skin lesions were generally higher in the MVA vaccinated group. With only 4 days between immunization and intravenous challenge, however, MVA still protected whereas Dryvax failed. Protection correlated with the more rapid immune response to MVA compared to Dryvax, which may be related to the higher dose of MVA that can be tolerated safely.
• biodefense
• modified vaccinia virus Ankara
• poxvirus
• smallpox vaccine
• neutralizing antibody
The world-wide smallpox eradication program sponsored by the WHO has been successful. Furthermore the legislation regarding smallpox vaccination in the Federal Republic of Germany has been given a new form. The place of the previous mass-vaccination has been taken by vaccination of individuals. Under this aspect safe procedures for vaccination are required. Any vaccination used by Public Health Service has to fulfill the following criteria: High protection against the disease without adverse side-effects or risks; it should be simple to administer and applicable under ordinary circumstances. For primary vaccination against smallpox the MVA-vaccination meets these demands entirely. It is reported about own experiences with 700 vaccinations using the attenuated virus "MVA"; complete documentation is given. Not a single complication nor any adverse side-effects have been seen. The Public Health Service has gained great experience in the field of vaccination. It is responsible for the protection of the individual as well as the public, as it is undesirable that there develops and unvaccinated group within the population. The individual claim a complete protection through vaccination which should be absolutely free of any risk.
A standard challenge with percutaneous smallpox vaccine was administered to 629 children six to 12 months after percutaneous primary inoculation with one of four vaccines (New York City Board of Health strains grown in calf lymph or chorioallantoic membranes, the Lister vaccine, or the CV-1 strain). Of those who had had major reactions on primary vaccination, 8%-21% responded to revasccination with a typical primary-type skin response. In contrast, such a primary-type response occurred in 50% of those who on primary vaccination had developed serum antibody in the absence of major reactions and in 83% of those who had had no serologic or clinical evidence of primary "take." Skin lesions on revaccination tended to be largest in thosewhose primary vaccination was with CV-1, although fever and minor complications were not more frequent. Moreover, even in children who had received CV-1 vaccine, skin responses to challenge vaccine were clearly attenuated when compared with responses of children who had not had takes on primary vaccination. Sizes of lesions and acceleration of skin erytherma after challenge were related in most children to titers of both hemagglutination-inhibiting and neutralizing antibody at the time of revaccination. One month after revaccination, neutralizing antibody was present in 93%-96% of those with takes onprimary vaccination with New York City or Lister vaccines, but only 75% of CV-1 vaccines.
The MVA virus is a lab virus ideally suited for vaccination of both man and animal which can be differentiated from the known Vaccinia strains by the use of numerous biological markers. Its reduced virulence for the chick embryo, for experimental animals and for man is a particularly characteristic feature. With the exception of chick embryo fibroblasts, the MVA virus grows in cell cultures only abortively. This applies particularly to cells of human origin in which the cytopathic effect and plaque formation are completely missing. The restriction analysis of the DNS of the MVA virus demonstrates that its genetic structure differs from that of the CVA basic virus and other orthopox viruses. In contrast to the WHO reference strain Elstree, the MVA virus has a genome shortened by about 9 per cent. The use of the MVA virus for human vaccination is particularly indicated in persons to be vaccinated for the first time and likely to entail a risk (on account of allergies etc.) because it brings about a state of revaccination without complications. The MVA virus can be administered in intracutaneous, subcutaneous or intramuscular injections. Innocuoursness and successful vaccination have been demonstrated in more than 120000 persons. While other Vaccinia strains, such as the Elstree virus, experience a drastic increase of virulence in the immunosuppressed organism (subjected to whole-body irradiation), the MVA virus cannot be activated not even in this situation.
In 1958, the Eleventh World Health Assembly, on the proposal of the USSR, approved a resolution for world-wide smallpox eradication. In that year alone the disease occurred in 59 countries, and in addition many other areas experienced imported cases. When the intensified eradication programme began in 1967, there were 33 countries with endemic smallpox and two more countries subsequently became endemic. These 33 countries had a population of over 1,200,000,000 and were located in four of the six WHO regions. The most important endemic area was in Asia, but the disease was also endemic in South America and in Africa, south of the Sahara. The intensified campaign quickly narrowed the endemic area. In April 1971, the last case was reported from Brazil, the stronghold of the disease in the Western Hemisphere. By 1973, with the exception of those in the Horn of Africa, smallpox transmission was interrupted in all African countries. The last case in Asia occurred in Bangladesh where variola major made its last stand. The last known case of smallpox in the world was reported from Somalia on 27 October 1977. If no more cases are discovered it will be possible, in two years from that date, to certify that smallpox has been eradicated from all areas of the world, and Member States of the World Health Organization will be able to celebrate an unprecedented victory for preventive medicine. The complete eradication of smallpox is not only the liberation of the world from one of its most dangerous diseases, but also provides an example of what can be achieved when countries throughout the world join together with a common aim.