[Show abstract][Hide abstract] ABSTRACT: Many viral infections, including HIV, exhibit sex-based pathogenic differences. However, few studies have examined vaccine-related sex differences. We compared immunogenicity and protective efficacy of monomeric SIV gp120 with oligomeric SIV gp140 in a pre-clinical rhesus macaque study and explored a subsequent sex bias in vaccine outcome. Each immunization group (16 females, 8 males) was primed twice mucosally with replication-competent Ad-recombinants encoding SIVsmH4env/rev, SIV239gag and SIV239nefΔ1-13 and boosted twice intramuscularly with SIVmac239 monomeric gp120 or oligomeric gp140 in MF59 adjuvant. Controls (7 females, 5 males) received empty Ad and MF59. Up to 9 weekly intrarectal challenges with low-dose SIVmac251 were administered until macaques became infected. We assessed vaccine-induced binding, neutralizing, and non-neutralizing antibodies, Env-specific memory B cells and plasmablasts/plasma cells (PB/PC) in bone marrow and rectal tissue, mucosal Env-specific antibodies, and Env-specific T-cells. Post-challenge, only one macaque (gp140-immunized) remained uninfected. However, SIV acquisition was significantly delayed in vaccinated females but not males, correlated with Env-specific IgA in rectal secretions, rectal Env-specific memory B cells, and PC in rectal tissue. These results extend previous correlations of mucosal antibodies and memory B cells with protective efficacy. The gp140 regimen was more immunogenic, stimulating elevated gp140 and cyclic V2 binding antibodies, ADCC and ADCP activities, bone marrow Env-specific PB/PC, and rectal gp140-specific IgG. However, immunization with gp120, the form of envelope immunogen used in RV144, the only vaccine trial to show some efficacy, provided more significant acquisition delay. Further over 40 weeks of follow-up, no gp120 immunized macaques met euthanasia criteria in contrast to 7 gp140-immunized and 2 control animals. Although males had higher binding antibodies than females, ADCC and ADCP activities were similar. The complex challenge outcomes may reflect differences in IgG subtypes, Fc glycosylation, Fc-R polymorphisms, and/or the microbiome, key areas for future studies. This first demonstration of a sex-difference in SIV vaccine-induced protection emphasizes the need for sex-balancing in vaccine trials. Our results highlight the importance of mucosal immunity and memory B cells at the SIV exposure site for protection.
[Show abstract][Hide abstract] ABSTRACT: Eliciting broadly reactive functional antibodies remains a challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development that is complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C, and novel antigenic properties have been described for subtype C Env proteins. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an infected person in the acute phase (Fiebig stage I/II) was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane-proximal external region (MPER), extended by a polylysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan blade" motifs when visualized by cryoelectron microscopy. CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16, and VRC01 HIV-1 neutralizing monoclonal antibodies (MAbs), as well as the V1/V2-specific PGT121, 697, 2158, and 2297 MAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers, and larger multimers elicited similar levels of crosssubtype binding and neutralizing antibodies to tier 1 and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus but did inhibit the homologous infectious molecular clone in a peripheral blood mononuclear cell (PBMC) assay. This Env is currently in good manufacturing practice (GMP) production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine.
Journal of Virology 05/2015; 89(15). DOI:10.1128/JVI.00412-15 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy.
[Show abstract][Hide abstract] ABSTRACT: HIV/SIV infection causes B-cell dysregulation and loss of memory B cells in PBMC. These effects are not completely reversed by anti-retroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cell/plasmablasts (PC/PB) in blood, bone marrow and lymph nodes of rhesus macaques during ART and upon release. Macaques previously immunized with SIV recombinants and gp120 protein were included to assess effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve, and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in activated memory and increases in tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27(+) or CD27(-) and therefore were defined as CD19(+)CD38(hi)CD138(+). These PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment allowing generation of viral-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.
Journal of Virology 09/2012; 86(23). DOI:10.1128/JVI.00298-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: We formerly constructed several DNA plasmids encoding HIV-1Ba-L gp160 containing point mutations and truncations in the cytoplasmic tail of gp41. In addition, we generated plasmid DNA encoding a V2/V3 mutant gp145 and the homologous, purified protein. We hypothesized that immunization of rabbits with these novel antigens, delivered in a DNA prime/protein boost regimen, would result in antibody responses of greater magnitude and breadth. We further hypothesized that immunization of rabbits, with a noted defect in B cell development and antibody selection, may give rise to neutralizing antibodies that would otherwise be removed due to their recognition of self proteins, lipids or long CDR3s. Methods: A rabbit immunogenicity study was performed to assess antibody responses elicited in New Zealand White (NZW) rabbits. For this, plasmid DNA encoding either gp160-Q708 (Q708) or gp145-V2/V3mut (V2/V3mut) was administered, by electroporation (Innovio), at weeks 0, 4 and 8. DNA-primed rabbits were then boosted, by intramuscular route, with ribi- (MPL, TDM, CWS)-adjuvanted recombinant protein (gp145-V2/V3mut) at weeks 22, 28 and 38. A similar schedule of DNA and protein immunizations was performed in an SLE (lupus) rabbit model system (a generous gift from Dr. Rose Mage, NIH). Binding antibody titers were assessed by ELISA and neutralizing antibody screens were conducted using a Tzm-b1 pseudo-virus assay. B-cell epitope mapping was performed, by pepscan ELISA, using 20mer peptides, overlapping by 14 amino acids. Results: NZW rabbits, immunized with plasmid DNA expressing Q708 or V2/V3mut, generated robust anti-gp145 titers following 3 administrations. Priming with Q708 yielded greater antibody titers (approx. 1 log) compared to V2/V3mut. Antibody titers in DNA-primed rabbits were further augmented following protein boost immunizations with adjuvanted-V2/V3mut protein. Following boost immunization, antibody titers were found to be comparable in both DNA-primed groups. By contrast, anti-gp145 antibody titers were found to be generally less in lupus rabbits following both DNA and protein immunizations. Antibody responses to 2F5/4E10 epitopes were detectable following DNA immunization of NZW rabbits in both (Q708, V2/V3mut) DNA-primed groups. Overall, antibody responses to 2F5/4E10 were comparable in lupus rabbits relative to that noted in NZW rabbits. Following protein boost immunization, 2F5/4E10 antibody responses were only found to be augmented in select rabbits (one from NZW group, one from lupus) that received the V2/V3mut homologous prime/boost regimen. Epitope mapping revealed that protein-boosted rabbits primed with Q708 DNA generated antibody responses to the C1, V2, V3, V5, C5 and immuno-dominant region of gp41. In contrast, protein-boosted rabbits primed with V2/V3mut DNA elicited antibody responses predominantly to the C1, V3, V5, C5 and HR2/MPER regions of gp41. Epitope responses were generally weaker in the V2/V3mut-primed group compared to the Q708-primed group. More robust antibody responses were found when immunizations were performed in lupus rabbits compared to NZW rabbits. In addition to epitope responses found in NZW rabbits, lupus rabbits generated additional antibody responses to the V1 and C4 regions (Q708-primed group), and against the V1, V2, C2/V3, C3, C4 and HR1 regions (V2/V3mut primed group). A neutralization screen found that the sera of immunized rabbits could neutralize a tier 1 (BaL. 1a) but not tier 2 (US1) viral isolate. Enhanced neutralization was not observed in lupus rabbits as compared to NZW rabbits. The greatest inhibition was noted in NZW rabbits primed with Q708 DNA and boosted with V2/V3mut protein. Conclusions: These findings suggest that a DNA prime/protein boost immunization strategy incorporating novel Env immunogens with c-tail truncations and V2/V3 loop mutations can elicit robust antibody responses that can neutralize a type-specific viral isolate. While increased breadth of epitope responses was acheived by immunizing lupus rabbits, increased neutralization was not observed against the Bal.1a or US1 isolate tested. Further studies are warranted to investigate if such changes incorporated into other clade B and clade C envelopes will increase binding and neutralizing antibody responses.
[Show abstract][Hide abstract] ABSTRACT: We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.
Journal of Virology 02/2012; 86(8):4644-57. DOI:10.1128/JVI.06812-11 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The characteristic event that follows infection of a cell by retroviruses Including human immunodeficiency virus (HIV)/ simian immunodeficiency virus (SIV) is the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Nuclear transport of newly synthesized viral DNA requires phosphorylation of proteins in the reverse transcription complex by virion-associated cellular kinases. Recently, we demonstrated that disruption of cellular mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 2 (ERK-2) incorporation into SIV virions inhibits virus replication in nonproliferating target cells, indicating that MAPK/ERK-2 plays an important role in HIV /SIV replication. The mechanism of incorporation of MAPK/ERK-2 into virus particles is not defined. In this regard, we hypothesized that a likely interaction of MAPK/ERK-2 with Gag(p55) may enable its packaging into virus particles. In the present investigation, we provided evidence for the first time that MAPK/ERK-2 interacts with the structural Gag polyprotein p55 using a combination of mutagenesis and protein-protein interaction analysis. We further show that MAPK/ERK-2 interacts specifically with the poly-proline motif present in the capsid region of Gag(p55). Utilizing virus-like particles directed by Gag, we have shown that the exchange of conserved proline residues within capsid of Gag(p55) resulted in impaired incorporation of MAPK/ERK-2. In addition, the deletion of a domain comprising amino acids 201 to 255 within host cell MAPK/ERK-2 abrogates its interaction with Gag(p55). The relevance of the poly-proline motif is further evident by its conservation in diverse retroviruses, as noted from the sequence analysis and structural modeling studies of predicted amino acid sequences of the corresponding Gag proteins. Collectively, these data suggest that the interaction of MAPK/ERK-2 with Gag polyprotein results in its incorporation into virus particles and may be essential for retroviral replication.
[Show abstract][Hide abstract] ABSTRACT: Three Indian rhesus macaques, Ad-SIV primed/protein boosted and exposed twice to high-dose mucosal SIV(mac251) challenges, exhibited elite control of viremia over 6.5 years. They were negative for host factors associated with control of SIV infection. After a third intrarectal challenge with SIV(smE660), all controlled viremia, with one (macaque #5) maintaining undetectable viremia in blood. Acquisition was not blocked, but virus was contained in the jejunum and draining lymph nodes. Polyfunctional memory T cell responses and high-titered neutralizing and non-neutralizing serum and mucosal antibodies were present before and maintained post-challenge. The level of protection seen for animal #5 was predicted from analyses of gene transcription in jejunum 2 weeks post-challenge. Macaques #7 and #9, exhibiting lower pre-challenge cellular and humoral immunity, partially controlled the SIV(smE660) challenge. Initial vaccine-induced control by macaque #5 extended to the SIV(smE660) challenge due to multiple immune mechanisms that were boosted and augmented by cryptic SIV exposure.
[Show abstract][Hide abstract] ABSTRACT: An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162) gp160 (subtype B) and HIV(TV1) gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIV(TV1) gp160 induced better cellular immunity than Ad5hr-HIV(SF162) gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIV(TV1) gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector.
[Show abstract][Hide abstract] ABSTRACT: Cell-mediated immunity and neutralizing antibodies contribute to control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but the role of nonneutralizing antibodies is not defined. Previously, we reported that sequential oral/oral or intranasal/oral (I/O) priming with replication-competent adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants, followed by intramuscular envelope protein boosting, elicited systemic and mucosal cellular immunity and exhibited equivalent, significant reductions of chronic viremia after rectal SIV(mac251) challenge. However, I/O priming gave significantly better control of acute viremia. Here, systemic and mucosal humoral immunity were investigated for potential correlates with the acute challenge outcome. Strong serum binding but nonneutralizing antibody responses against SIV(mac251) were induced in both groups. Antibody responses appeared earlier and overall were higher in the I/O group. Reduced acute viremia was significantly correlated with higher serum binding titer, stronger antibody-dependent cellular cytotoxicity activity, and peak prechallenge and 2-week-postchallenge antibody-dependent cell-mediated viral inhibition (ADCVI). The I/O group consistently displayed greater anti-envelope immunoglobulin A (IgA) antibody responses in bronchoalveolar lavage and a stronger rectal anti-envelope IgA anamnestic response 2 weeks postchallenge. Pre- and postchallenge rectal secretions inhibited SIV transcytosis across epithelial cells. The inhibition was significantly higher in the I/O group, although a significant correlation with reduced acute viremia was not reached. Overall, the replicating Ad5hr-SIV priming/envelope boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities. The pattern of elevated immune responses in the I/O group is consistent with its better control of acute viremia mediated, at least in part, by ADCVI activity and transcytosis inhibition.
Journal of Virology 11/2008; 83(2):791-801. DOI:10.1128/JVI.01672-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.
Journal of Virology 09/2008; 82(21):10911-21. DOI:10.1128/JVI.01129-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.