The vast diversity of HIV-1 infections has greatly impeded the development of a successful HIV-1/AIDS vaccine. Previous vaccine work has demonstrated limited levels of protection against SHIV/SIV infection, but protection was observed only when the challenge virus was directly matched to the vaccine strain. As it is likely impossible to directly match the vaccine strain to all infecting strains in nature, it is necessary to develop an HIV-1 vaccine that can protect against a heterologous viral challenge. In this study we investigated the ability of polyvalent and consensus vaccines to protect against a heterologous clade B challenge. Rhesus macaques were vaccinated with ConB or PolyB virus-like particle vaccines. All vaccines were highly immunogenic with high titers of antibody found in all vaccinated groups against SIV Gag. Antibody responses were also observed against a diverse panel of clade B envelopes. Following vaccination nonhuman primates (NHPs) were challenged via the vaginal route with SHIVSF162p4. The PolyB vaccine induced a 66.7% reduction in the rate of infection as well as causing a two log reduction in viral burden if infection was not blocked. ConB vaccination had no effect on either the infection rate or viral burden. These results indicate that a polyvalent clade-matched vaccine is better able to protect against a heterologous challenge as compared to a consensus vaccine.
Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production.
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.
Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.
Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.
Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.
We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.
We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.
Salicylidene acylhydrazides; HIV; microbicide; iron chelation
We reported previously that while prolonged tenofovir monotherapy of macaques infected with virulent simian immunodeficiency virus (SIV) resulted invariably in the emergence of viral mutants with reduced in vitro drug susceptibility and a K65R mutation in reverse transcriptase, some animals controlled virus replication for years. Transient CD8+ cell depletion or short-term tenofovir interruption within 1 to 5 years of treatment demonstrated that a combination of CD8+ cell-mediated immune responses and continued tenofovir therapy was required for sustained suppression of viremia. We report here follow-up data on 5 such animals that received tenofovir for 8 to 14 years.
Although one animal had a gradual increase in viremia from 3 years onwards, the other 4 tenofovir-treated animals maintained undetectable viremia with occasional viral blips (≤ 300 RNA copies/ml plasma). When tenofovir was withdrawn after 8 to 10 years from three animals with undetectable viremia, the pattern of occasional episodes of low viremia (≤ 3600 RNA/ml plasma) continued throughout the 10-month follow-up period. These animals had low virus levels in lymphoid tissues, and evidence of multiple SIV-specific immune responses.
Under certain conditions (i.e., prolonged antiviral therapy initiated early after infection; viral mutants with reduced drug susceptibility) a virus-host balance characterized by strong immunologic control of virus replication can be achieved. Although further research is needed to translate these findings into clinical applications, these observations provide hope for a functional cure of HIV infection via immunotherapeutic strategies that boost antiviral immunity and reduce the need for continuous antiretroviral therapy.
Tenofovir; PMPA; SIV; Functional cure; Antiretroviral; HIV
Maternal HIV-1-specific antibodies are efficiently transferred to newborns; their role in disease control is unknown. We administered non-sterilizing levels of neutralizing IgG, including the human neutralizing monoclonal IgG1b12, to six newborn macaques before oral challenge with SHIVSF612P3. All rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for 6 months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings.
Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.
The membrane-proximal external region (MPER) of HIV-1, located at the C terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10, and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal simian-human immunodeficiency virus (SHIV) challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of six male Indian rhesus macaques 1 day prior to and again 1 day following intrarectal challenge with SHIVBa-L. In both groups, five out of six animals showed complete protection and sterilizing immunity, while for one animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.
Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for human immunodeficiency virus type 1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing simian immunodeficiency virus (SIV) Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, single-shot immunization may ultimately be preferable for global vaccine delivery. We therefore evaluated the immunogenicity and protective efficacy of a single immunization of chimeric rAd5 hexon hypervariable region 48 (rAd5HVR48) vectors expressing SIV Gag, Pol, Nef, and Env against a homologous SIV challenge in rhesus monkeys. Inclusion of Env resulted in improved control of peak and set point SIV RNA levels following challenge. In contrast, DNA vaccine priming did not further improve the protective efficacy of rAd5HVR48 vectors in this system.
A recombinant adenovirus serotype 5 (rAd5) vector-based vaccine for HIV-1 has recently failed in a phase 2b efficacy study in humans1, 2. Consistent with these results, preclinical studies have demonstrated that rAd5 vectors expressing SIV Gag failed to reduce peak or setpoint viral loads following SIV challenge of rhesus monkeys that lacked the protective MHC class I allele Mamu-A*013. Here we show that an improved T cell-based vaccine regimen utilizing two serologically distinct adenovirus vectors afforded substantially improved protective efficacy in this stringent challenge model. In particular, a heterologous rAd26 prime, rAd5 boost vaccine regimen expressing SIV Gag elicited cellular immune responses with augmented magnitude, breadth, and polyfunctionality as compared with the homologous rAd5 regimen. Following SIVmac251 challenge, monkeys vaccinated with the heterologous rAd26/rAd5 regimen exhibited a 1.4 log reduction of peak and a 2.4 log reduction of setpoint viral loads as well as decreased AIDS-related mortality as compared with control animals. These data demonstrate that durable partial immune control of a pathogenic SIV challenge for over 500 days can be achieved by a T cell-based vaccine in Mamu-A*01-negative rhesus monkeys in the absence of a homologous Env antigen. These findings have important implications for the development of next generation T cell-based vaccine candidates for HIV-1.
A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.
Developing an immunogen that elicits broadly neutralizing antibodies (bNAbs) is an elusive but important goal of HIV vaccine research, especially after the recent failure of the leading T cell based HIV vaccine in human efficacy trials. Even if such an immunogen can be developed, most animal model studies indicate that high serum neutralizing concentrations of bNAbs are required to provide significant benefit in typical protection experiments. One possible exception is provided by the anti-glycan bNAb 2G12, which has been reported to protect macaques against CXCR4-using SHIV challenge at relatively low serum neutralizing titers. Here, we investigated the ability of 2G12 administered intravenously (i.v.) to protect against vaginal challenge of rhesus macaques with the CCR5-using SHIVSF162P3. The results show that, at 2G12 serum neutralizing titers of the order of 1∶1 (IC90), 3/5 antibody-treated animals were protected with sterilizing immunity, i.e. no detectable virus replication following challenge; one animal showed a delayed and lowered primary viremia and the other animal showed a course of infection similar to 4 control animals. This result contrasts strongly with the typically high titers observed for protection by other neutralizing antibodies, including the bNAb b12. We compared b12 and 2G12 for characteristics that might explain the differences in protective ability relative to neutralizing activity. We found no evidence to suggest that 2G12 transudation to the vaginal surface was significantly superior to b12. We also observed that the ability of 2G12 to inhibit virus replication in target cells through antibody-mediated effector cell activity in vitro was equivalent or inferior to b12. The results raise the possibility that some epitopes on HIV may be better vaccine targets than others and support targeting the glycan shield of the envelope.
An effective HIV vaccine should elicit broadly neutralizing antibodies, i.e. antibodies that neutralize a wide spectrum of different HIVs in vitro. A number of human monoclonal antibodies have been isolated with broad neutralization and shown to protect macaques against vaginal HIV challenge. Protection is generally correlated with neutralization and requires relatively high antibody concentrations that may be difficult to achieve by vaccination. Here, we show that one monoclonal antibody (2G12) is unusually potent in protection relative to its neutralizing ability as hinted at by earlier data. Further studies eliminate an unusual ability of 2G12 to be transported to the vagina (site of infection) as a possible explanation for our observations. Although the precise mechanism is unclear, the studies have important implications for HIV vaccine design in general by suggesting that some vaccine targets on HIV may be better than others and, specifically, by suggesting that the sugar coat of HIV may be a particularly rewarding target if appropriate immunogens can be designed.
Lentiviruses such as human and simian immunodeficiency viruses (HIV and SIV) undergo continual evolution in the host. Previous studies showed that the late-stage variants of SIV that evolve in one host replicate to significantly higher levels when transmitted to a new host. However, it is unknown whether HIVs or SIVs that have higher replication fitness are more genetically stable upon transmission to a new host. To begin to address this, we analyzed the envelope sequence variation of viruses that evolved in animals infected with variants of SIVMne that had been cloned from an index animal at different stages of infection.
We found that there was more evolution of envelope sequences from animals infected with the late-stage, highly replicating variants than in animals infected with the early-stage, lower replicating variant, despite the fact that the late virus had already diversified considerably from the early virus in the first host, prior to transmission. Many of the changes led to the addition or shift in potential-glycosylation sites-, and surprisingly, these changes emerged in some cases prior to the detection of neutralizing antibody responses, suggesting that other selection mechanisms may be important in driving virus evolution. Interestingly, these changes occurred after the development of antibody whose anti-viral function is dependent on Fc-Fcγ receptor interactions.
SIV variants that had achieved high replication fitness and escape from neutralizing antibodies in one host continued to evolve upon transmission to a new host. Selection for viral variants with glycosylation and other envelope changes may have been driven by both neutralizing and Fcγ receptor-mediated antibody activities.
Although antibodies can prevent or modulate lentivirus infections in nonhuman primates, the biological functions of antibody responsible for such effects are not known. We sought to determine the role of antibody-dependent cell-mediated virus inhibition (ADCVI), an antibody function that inhibits virus yield from infected cells in the presence of Fc receptor-bearing effector cells, in preventing or controlling SIVmac251 infection in rhesus macaques (Macaca mulatta). Using CEMx174 cells infected with simian immunodeficiency virus mac251 (SIVmac251), both polyclonal and monoclonal anti-SIV antibodies were capable of potent virus inhibition in the presence of human peripheral blood mononuclear cell (PBMC) effector cells. In the absence of effector cells, virus inhibition was generally very poor. PBMCs from healthy rhesus macaques were also capable of mediating virus inhibition either against SIVmac251-infected CEMx174 cells or against infected, autologous rhesus target cells. We identified both CD14+ cells and, to a lesser extent, CD8+ cells as the effector cell population in the rhesus PBMCs. Finally, pooled, nonneutralizing SIV-antibody-positive serum, shown in a previous study to prevent infection of neonatal macaques after oral SIVmac251 challenge, had potent virus-inhibitory activity in the presence of effector cells; intact immunoglobulin G, rather than F(ab′)2, was required for such activity. This is the first demonstration of both humoral and cellular ADCVI functions in the macaque-SIV model. ADCVI activity in nonneutralizing serum that prevents SIV infection suggests that ADCVI may be a protective immune function. Finally, our data underscore the potential importance of Fc-Fc receptor interactions in mediating biological activities of antibody.
Infection of macaques with the avirulent molecular clone SIVmac1A11 results in transient low viremia and no disease. To investigate if this low viremia is solely due to intrinsic poor replication fitness or is mediated by efficient immune-mediated control, 5 macaques were inoculated intravenously with SIVmac1A11. Three animals that were depleted of CD8+ cells at the start of infection had more prolonged viremia with peak virus levels 1 to 2 logs higher than those of 2 animals that received a non-depleting control antibody. Thus, CD8+ cell-mediated immune responses play an important role in controlling SIVmac1A11 replication during acute viremia.
Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4+ lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcγRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4+ lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab′)2 was used. Further experiments demonstrated that the release of β-chemokines, most likely through FcγR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcγR interactions enhance the ability of antibody to neutralize HIV-1. Since FcγR-bearing cells are always present in vivo, FcγR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.
The partial control of viremia during acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by an HIV-1-specific cytotoxic T-lymphocyte (CTL) response and an absent or infrequent neutralizing antibody response. The control of HIV-1 viremia has thus been attributed primarily, if not exclusively, to CTL activity. In this study, the role of antibody in controlling viremia was investigated by measuring the ability of plasma or immunoglobulin G from acutely infected patients to inhibit primary strains of HIV-1 in the presence of natural-killer (NK) effector cells. Antibody that inhibits virus when combined with effector cells was present in the majority of patients within days or weeks after onset of symptoms of acute infection. Furthermore, the magnitude of this effector cell-mediated antiviral antibody response was inversely associated with plasma viremia level, and both autologous and heterologous HIV-1 strains were inhibited. Finally, antibody from acutely infected patients likely reduced HIV-1 yield in vitro both by mediating effector cell lysis of target cells expressing HIV-1 glycoproteins and by augmenting the release of β-chemokines from NK cells. HIV-1-specific antibody may be an important contributor to the early control of HIV viremia.
In some countries, excessive non-measles-related mortality has been observed among female recipients of high-titer measles vaccines. We determined if differences in the immune response to measles vaccines underlie the excessive female mortality by measuring the measles virus (MV)-specific antibody-dependent cellular cytotoxicity (ADCC) antibody response in 65 3-year-old Gambian children immunized with Edmonston-Zagreb medium-titer (EZ) or Schwarz standard vaccines during infancy. Among the 20 females and 22 males with undetectable anti-MV antibodies at the time of immunization, females had significantly lower ADCC than males (median cytotoxicities of 1/100 serum dilutions = 8.4 and 12%, respectively; P = 0.04). This sex-associated difference was present only among the six female and seven male recipients of EZ vaccine (median cytotoxicities = 5.1 and 19.0%, respectively; P = 0.02). There were no significant sex-associated differences in neutralizing antibody activity. Decreased ADCC antibody activity may contribute to the lower survival rate observed in females receiving high-titer measles vaccination.