We previously showed that expression of human FcγRI on TZM-bl cells potentiates neutralization by gp41 membrane-proximal external region (MPER)-specific antibodies. Here we show that lysosomotropic reagents known to block phagocytosis do not diminish this effect. We also show that FcγRI occasionally potentiates neutralization by antibodies against the V3 loop of gp120 and cluster I of gp41. We conclude that FcγRI provides a kinetic advantage for neutralizing antibodies against partially cryptic epitopes independent of phagocytosis.
Sensitive assays are needed to meaningfully assess low levels of neutralizing antibodies (NAbs) that may be important for protection against the acquisition of HIV-1 infection in vaccine recipients. The current assay of choice uses a non-lymphoid cell line (TZM-bl) that may lack sensitivity owing to over expression of CD4 and CCR5. We used transfection of a human CD4+/CXCR4+/α4β7+ T-lymphoblastoid cell line (A3.01) with a CMV IE promoter-driven CCR5neo vector to stably express CCR5. The resulting line, designated A3R5, is permissive to a wide range of CCR5-tropic circulating strains of HIV-1, including HIV-1 molecular clones containing a Tat-inducible Renilla luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens.
Intramuscular injection of macaques with an IL-12 expression plasmid (0.1 or 0.4 mg DNA/animal) optimized for high level of expression and delivered using in vivo electroporation, resulted in the detection of systemic IL-12 cytokine in the plasma. Peak levels obtained by day 4–5 post injection were paralleled by a rapid increase of IFN-γ, indicating bioactivity of the IL-12 cytokine. Both plasma IL-12 and IFN-γ levels were reduced to basal levels by day 14, indicating a short presence of elevated levels of the bioactive IL-12. The effect of IL-12 as adjuvant together with an SIVmac239 DNA vaccine was further examined comparing two groups of rhesus macaques vaccinated in the presence or absence of IL-12 DNA. The IL-12 DNA-adjuvanted group developed significantly higher SIV-specific cellular immune responses, including IFN-γ+ Granzyme B+ T cells, demonstrating increased levels of vaccine-induced T cells with cytotoxic potential, and this difference persisted for 6 mo after the last vaccination. Coinjection of IL-12 DNA led to increases in Gag-specific CD4+ and CD4+CD8+ double-positive memory T cell subsets, whereas the Env-specific increases were mainly mediated by the CD8+ and CD4+CD8+ double-positive memory T cell subsets. The IL-12 DNA-adjuvanted vaccine group developed higher binding antibody titers to Gag and mac251 Env, and showed higher and more durable neutralizing antibodies to heterologous SIVsmE660. Therefore, co-delivery of IL-12 DNA with the SIV DNA vaccine enhanced the magnitude and breadth of immune responses in immunized rhesus macaques, and supports the inclusion of IL-12 DNA as vaccine adjuvant.
in vivo electroporation; HIV; SIVsmE660; SIVmac239; antibody; neutralizing antibody; avidity; central memory; transitional memory; effector memory
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in ~20% of HIV-1-infected individuals, and details of their generation could provide a roadmap for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection. The mature antibody, CH103, neutralized ~55% of HIV-1 isolates, and its co-crystal structure with gp120 revealed a novel loop-based mechanism of CD4-binding site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the CH103-lineage unmutated common ancestor avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data elucidate the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies and provide insights into strategies to elicit similar antibodies via vaccination.
A comparative evaluation of the immunity stimulated with a vaccine regimen that includes simian immunodeficiency virus (SIV), interleukin 2 (IL-2), and IL-15 DNAs, recombinant modified vaccinia virus Ankara (rMVA), and inactivated SIVmac239 particles administered into the oral and nasal cavities, small intestine, and vagina was carried out in female rhesus macaques to determine the best route to induce diverse anti-SIV immunity that may be critical to protection from SIV infection and disease. All four immunizations generated mucosal SIV-specific IgA. Oral immunization was as effective as vaginal immunization in inducing SIV-specific IgA in vaginal secretions and generated greater IgA responses in rectal secretions and saliva samples compared to the other immunization routes. All four immunizations stimulated systemic T-cell responses against Gag and Env, albeit to a different extent, with oral immunization providing greater magnitude and nasal immunization providing wider functional heterogeneity. SIV-specific T cells producing gamma interferon (IFN-γ) dominated these responses. Limited levels of SIV-specific IgG antibodies were detected in plasma samples, and no SIV-specific IgG antibodies were detected in secretions. Vaccination also induced CD4+ and CD8+ T-cell responses in the rectal and vaginal mucosa with greater functional heterogeneity than in blood samples. Rectal T-cell responses were significantly greater in the orally vaccinated animals than in the other animals. The most balanced, diverse, and higher-magnitude vaginal T-cell responses were observed after intestinal vaccination. Significantly higher CD8+ granzyme B-positive T-cell responses were observed systemically after intestinal vaccination and in rectal cells after oral immunization. The majority of SIV-specific T cells that produced granzyme B did not produce cytokines. Of the immunization routes tested, oral vaccination provided the most diverse and significant response to the vaccine.
The development of a vaccine against human immunodeficiency virus-1 (HIV-1) capable of inducing broad humoral and cellular responses at both the systemic and mucosal levels will be critical for combating the global AIDS epidemic. We previously demonstrated the ability of Newcastle disease virus (NDV) as a vaccine vector to express oligomeric Env protein gp160 and induce potent humoral and mucosal immune responses. In the present study, we used NDV vaccine strain LaSota as a vector to compare the biochemical and immunogenic properties of vector-expressed gp160, gp120, and two versions of gp140 (a derivative of gp160 made by deleting the transmembrane and cytoplasmic domains), namely: gp140L, which contained the complete membrane-proximal external region (MPER), and gp140S, which lacks the distal half of MPER. We show that, similar to gp160, NDV-expressed gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Immunization with rLaSota/140S, rLaSota/140L rLaSota/120 resulted in mixed Th1/Th2 immune responses as compared to Th1-biased immune responses induced by rLaSota/160. Importantly, rLaSota/gp140S induced neutralizing antibody responses to homologous HIV-1 strain BaL.26 and laboratory adapted HIV-1 strain MN.3 that were stronger than those elicited by the other NDV recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses in mice. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein.
To characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque (RM), RAt-9, which had been challenged sequentially with two related clade C simian-human immunodeficiency viruses (SHIV-Cs) yet remained aviremic for >5 years despite indirect evidence of cryptic infection.
To measure long-term anti-SHIV-C immunity, host genetics and gene-expression patterns for protective correlates.
Long-term immune reactivity was evaluated and identification of virus in RAt-9 was attempted by RT-PCR analysis of concentrated plasma and blood transfer to CD8+ cell-depleted infant macaques. Full MHC genotyping of RAt-9, TRIM5α and KIR3DL allelic expression analysis of PBMC, and microarray gene expression analysis were performed.
All attempts to detect/isolate virus, including blood transfer to CD8+ cell-depleted infant RM, were negative, and the animal maintained normal levels of memory CD4+ T cells in both peripheral blood and gut tissues. However, RAt-9 maintained high levels of anti-SHIV-C humoral and cellular immunity, including reactivity to non-vaccine neoantigens (Nef and Rev), up to 63 months post-initial challenge, suggesting chronic sub-threshold infection. RAt-9 expressed the Mamu A*001 allele but was B*008−B*017−, had a B13 serotype, and had increased expression of killer-cell immunoglobulin-like receptors (KIRs) previously linked to favorable outcomes of lentiviral infection. Elements of the gene expression profiling coincided with genotyping results. RAt-9 also displayed CD8+ cell noncytotoxic antiviral response (CNAR) activity.
RAt-9 is the first example of a virus-exposed, persistently aviremic animal that has maintained long-term, high-level cellular and humoral antiviral immunity in the absence of an identifiable cryptic reservoir.
AIDS; vaccine; SHIV; clade C; long-term immunity; prevention of viremia
An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of human immunodeficiency virus type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime–protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial. Robust cross-subtype HIV-1-specific T cell responses were detected in IFN-γ ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime–protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.
HIV-1; DNA vaccine; Recombinant protein vaccine; Phase I clinical trial; Prime–boost
The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was an about one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanism of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.
We have developed a high throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6±2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.
ADCC; HIV; SIV; NK; Fc gamma receptors; Granzyme B; high throughput
In a previous vaccine study, we reported significant and apparently sterilizing immunity to high-dose, mucosal, simian immunodeficiency virus (SIV) quasispecies challenge (27). The vaccine consisted of vectors based on vesicular stomatitis virus (VSV) expressing simian immunodeficiency virus (SIV) gag and env genes, a boost with propagating replicon particles expressing the same SIV genes, and a second boost with VSV-based vectors. Concurrent with that published study we had a parallel group of macaques given the same doses of vaccine vectors, but in addition, we included a third VSV vector expressing rhesus macaque GM-CSF in the priming immunization only. We report here that addition of the vector expressing GM-CSF did not enhance CD8 T cell or antibody responses to SIV antigens, and almost completely abolished the vaccine protection against high-dose mucosal challenge with SIV. Expression of GM-CSF may have limited vector replication excessively in the macaque model. Our results suggest caution in the use of GM-CSF as a vaccine adjuvant, especially when expressed by a viral vector. Combining vaccine group animals from this study and the previous study we found that there was a marginal but significant positive correlation between the neutralizing antibody to a neutralization resistant SIV Env and protection from infection.
To determine the spectrum of antiviral antibodies in HIV-1-infected individuals in whom viral replication is spontaneously undetectable, termed HIV controllers (HICs).
Multicenter French trial ANRS EP36 studying the viral control in HICs.
Neutralizing Antibody (nAb) activities (neutralization assay, competition with broadly reactive monoclonal antibodies, and reactivity against the viral MPER gp41 region), FcγR-mediated antiviral activities, antibody-dependent cell cytotoxicity (ADCC), as well as autoantibody levels, were quantified in plasma from 22 controllers and from viremic individuals. The levels of these different antibody responses and HIV-specific CD8 T cell responses quantified by enzyme-linked immunosorbent spot (ELISPOT) IFNγ assay were compared in each controller.
The levels of antibody against the gp120 CD4 binding site, gp41, as well as Env epitopes near to the sites bound by broadly nAbs 2F5 and 1b12 were not different between HICs and viremic individuals. We did not find significant autoantibody levels in HICs. The magnitude and breadth of nAbs were heterogeneous in HICs but lower than in viremic individuals. The levels of nAbs using FcγR-mediated assay inhibition were similar in both groups. Regardless of the type of antibody tested, there was no correlation with HIV-specific CD8 T cell responses. ADCC was detectable in all controllers tested and was significantly higher than in viremic individuals (P <0.0002).
There was no single anti-HIV-1 antibody specificity that was a clear correlate of immunity in controllers. Rather, for most antibody types, controllers had the same or lower levels of nAbs than viremic individuals, with the possible exception of ADCC antibodies.
antibody-dependent cell cytotoxicity; FcγR; HIV controller; humoral immunity; neutralizing antibodies
Broadly neutralizing antibodies to the CD4 binding site (CD4bs) of gp120 are generated by some HIV-1-infected individuals, but little is known about the prevalence and evolution of this antibody response during the course of HIV-1 infection. We analyzed the sera of 113 HIV-1 seroconverters from three cohorts for binding to a panel of gp120 core proteins and their corresponding CD4bs knockout mutants. Among sera collected between 99 and 258 weeks post-HIV-1 infection, 88% contained antibodies to the CD4bs and 47% contained antibodies to resurfaced stabilized core (RSC) probes that react preferentially with broadly neutralizing CD4bs antibodies (BNCD4), such as monoclonal antibodies (MAbs) VRC01 and VRC-CH31. Analysis of longitudinal serum samples from a subset of 18 subjects revealed that CD4bs antibodies to gp120 arose within the first 4 to 16 weeks of infection, while the development of RSC-reactive antibodies was more varied, occurring between 10 and 152 weeks post-HIV-1 infection. Despite the presence of these antibodies, serum neutralization mediated by RSC-reactive antibodies was detected in sera from only a few donors infected for more than 3 years. Thus, CD4bs antibodies that bind a VRC01-like epitope are often induced during HIV-1 infection, but the level and potency required to mediate serum neutralization may take years to develop. An improved understanding of the immunological factors associated with the development and maturation of neutralizing CD4bs antibodies during HIV-1 infection may provide insights into the requirements for eliciting this response by vaccination.
Vaccine regimens using different agents for priming and boosting have become popular for enhancing T cell and Ab responses elicited by candidate HIV/AIDS vaccines. Here we use a simian model to evaluate immunogenicity and protective efficacy of a recombinant modified vaccinia Ankara (MVA) vaccine in the presence and absence of a recombinant DNA prime. The simian vaccines and regimens represent prototypes for candidate HIV vaccines currently undergoing clinical testing.
Recombinant DNA and MVA immunogens expressed simian immunodeficiency virus (SIV)mac239 Gag, PR, RT, and Env sequences. Vaccine schedules tested inoculations of MVA at months 0, 2, and 6 (MMM regimen) or priming with DNA at months 0 and 2 and boosting with MVA at months 4 and 6 (DDMM regimen). Twelve weekly rectal challenges with the heterologous SIV smE660 were initiated at 6 months following the last immunization.
Both regimens elicited similar 61–64% reductions in the per challenge risk of SIVsmE660 transmission despite raising different patterns of immune responses. The DDMM regimen elicited higher magnitudes of CD4 T cells whereas the MMM regimen elicited higher titers and greater avidity Env-specific IgG and more frequent and higher titer SIV-specific IgA in rectal secretions. Both regimens elicited similar magnitudes of CD8 T cells. Magnitudes of T cell responses, specific activities of rectal IgA Ab, and the tested specificities for neutralization and antibody-dependent cellular cytotoxicity did not correlate with risk of infection. However, the avidity of Env-specific IgG had a strong correlation with the per challenge risk of acquisition, but only for the DDMM group.
We conclude that for the tested immunogens in rhesus macaques, the simpler MMM regimen is as protective as the more complex DDMM regimen.
Vaccine; Immunodeficiency virus; Simian immunodeficiency virus; DNA vaccine MVA vaccine; avidity in protection
There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in
vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.
The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.
Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.
The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.
We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.
Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs.
Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy.
The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV89.6P replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as two weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by four weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R = -0.83, p 0.015), and ADCVI (R = -0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV89.6P variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV89.6 and virus levels (R = -0.72 p =0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV89.6P challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing FcγR-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.
Purpose of review
New findings continue to support the notion that broadly crossreactive neutralizing antibody induction is a worthwhile and achievable goal for HIV-1 vaccines. Immunogens are needed that can overcome the genetic variability and complex immune evasion tactics of the virus. Other antibodies might bridge innate and acquired immunity for possible beneficial vaccine effects. This review summarizes progress made over the past year that has enhanced our understanding of humoral immunity as it relates to HIV-1 vaccine development.
Although a clear path to designing an effective neutralizing antibody-based HIV-1 vaccine remains elusive, there is new information on how antibodies neutralize HIV-1, the epitopes involved, and clues to the possible nature of protective immunogens that keep this goal alive. Moreover, there is a greater understanding of HIV-1 diversity and its possible limits under immune pressure. Other antibodies might possess antiviral activity by mechanisms involving Fc receptor engagement or complement activation that would be of value for HIV-1 vaccines.
Recent developments strengthen the rationale for antibody-based HIV-1 vaccine immunogens and provide a stronger foundation for vaccine discovery.
adjuvants; AIDS; antibodies; complement; Fc receptors; vaccines
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
Plasma from a small subset of subjects chronically infected with HIV-1 shows remarkable magnitude and breadth of neutralizing activity. From one of these individuals (CH0219), we isolated two broadly neutralizing antibodies (bnAbs), CH01 and VRC-CH31, from two clonal lineages of memory B cells with distinct specificities (variable loop 1 and 2 [V1V2] conformational specificity and CD4-binding site specificity, respectively) that recapitulate 95% of CH0219 serum neutralization breadth. These data provide proof of concept for an HIV-1 vaccine that aims to elicit bnAbs of multiple specificities.
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.
The influence of preexisting immunity to viral vectors is a major issue for the development of viral vectored vaccines. Here, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/MVA SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses, but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of a SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively in preimmune animals compared to control animals. The peak and set point viremia correlated directly with colorectal virus at 2 weeks post challenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included (i) the presence of anti-SIV IgA in rectal secretions, (ii) high avidity binding antibody for the native form of Env and (iii) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral co-receptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 weeks post challenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.