Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological significance of this epitope for the virus.     

Correlation between env V1/V2 region diversification and neutralizing antibodies during primary infection by simian immunodeficiency virus sm in rhesus macaques

Evolution of the domain encoding the V1/V2 variable region of the simian immunodeficiency virus sm (SIVsm) envelope (env) gene was analyzed in relation to route of virus challenge, virus load, and neutralizing antibody (NAb) titers during primary infection of rhesus macaques with the pathogenic SIVsmE660 isolate. In this model system animals are initially infected with multiple viruses as evidenced by the presence of multiple V1/V2 genotypic variants that could be resolved by using a heteroduplex tracking assay (HTA). Overlapping subsets of the multiple variants were established in each animal. There was no selection for the establishment of specific variants in comparing intravenous- and intrarectal-challenged macaques at week 2 postinfection, suggesting that no genotypic selection occurred at the mucosal surface. There was an initial period of significant stability of the V1/V2 variants. Macaques challenged intravenously displayed subsequent V1/V2 diversification significantly earlier than macaques challenged intrarectally and well past the initial resolution of viremia. The time when SIVsmE660-specific NAbs reached a threshold titer of 100 was significantly correlated with the timing of V1/V2 diversification, even though antibodies to the Env protein could be detected much earlier. The time when NAbs reached a titer of 400 was significantly correlated with virus load late in infection. These results show that the route of infection affects the timing of V1/V2 diversification and that this diversification is correlated with the maturation of a specific NAb response. However, prior immunization capable of priming an anamnestic Env antibody response did not accelerate V1/V2 diversification. This result suggests that diversification of the SIV env V1/V2 region is the result of a type-specific antibody response.     

Potent antibody-mediated neutralization and evolution of antigenic escape variants of simian immunodeficiency virus strain SIVmac239 in vivo

Here, we describe the evolution of antigenic escape variants in a rhesus macaque that developed unusually high neutralizing antibody titers to SIVmac239. By 42 weeks postinfection, 50% neutralization of SIVmac239 was achieved with plasma dilutions of 1:1,000. Testing of purified immunoglobulin confirmed that the neutralizing activity was antibody mediated. Despite the potency of the neutralizing antibody response, the animal displayed a typical viral load profile and progressed to terminal AIDS with a normal time course. Viral envelope sequences from week 16 and week 42 plasma contained an excess of nonsynonymous substitutions, predominantly in V1 and V4, including individual sites with ratios of nonsynonymous to synonymous substitution rates (dN/dS) highly suggestive of strong positive selection. Recombinant viruses encoding envelope sequences isolated from these time points remained resistant to neutralization by all longitudinal plasma samples, revealing the failure of the animal to mount secondary responses to the escaped variants. Substitutions at two sites with significant dN/dS values, one in V1 and one in V4, were independently sufficient to confer nearly complete resistance to neutralization. Substitutions at three additional sites, one in V4 and two in gp41, conferred moderate to high levels of resistance when tested individually. All the amino acid changes leading to escape resulted from single nucleotide substitutions. The observation that antigenic escape resulted from individual, single amino acid replacements at sites well separated in current structural models of Env indicates that the virus can utilize multiple independent pathways to rapidly achieve similar levels of resistance.     

Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways.     

Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies.

During progression to AIDS in simian immunodeficiency virus (SIV) Mne-infected macaques, viral variants are selected that encode sequences with serine and threonine changes in variable region 1 (V1) of the surface component of the viral envelope protein (Env-SU). Because these serine and threonine amino acid changes are characteristic of sites for O-linked and N-linked glycosylation, we examined whether they were targets for modification by carbohydrates. For this purpose, we used several biochemical methods for analyzing the Env-SU protein encoded by chimeras of SIVMneCL8 and envelope sequences cloned from an SIVMneCL8-infected Macaca nemestrina during clinical latency and just after the onset of AIDS. The addition of an N-linked glycan was demonstrated by changes in the electrophoretic mobility of Env-SU, and this was verified by specific glycanase digestions and a detailed analysis of the molecular mass of partially purified Env-SU by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Molecular mass calculations by MALDI-TOF MS also demonstrated an increased mass, from 102.3 to 103.5 kDa, associated with serine and threonine residues predicted to be O-linked glycosylation sites. Together, these data provide the first direct evidence that the carbohydrate profile of Env-SU is distinct in SIV variants that evolve during infection of the host. Moreover, our studies show that these changes in glycosylation in V1 were directly associated with changes in antigenicity. Specifically, serine and threonine changes in V1 allowed the virus to escape neutralization by macaque sera that contained antibodies that could neutralize the parental virus, SIVMneCL8. The escape from antibody recognition appeared to be influenced by either O-linked or N-linked carbohydrate additions in V1. Moreover, when glycine residues were engineered at the positions where serine and threonine changes evolve in V1 of SIVMneCL8, there was no change in antigenicity compared to SIVMneCL8. This suggests that the amino acids in V1 are not part of the linear epitope recognized by neutralizing antibody. More likely, V1-associated carbohydrates mask the major neutralizing epitope of SIV. These experiments indicate that the selection of novel glycosylation sites in the V1 region of envelope during the course of disease is driven by humoral immune responses.     

Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV.     

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

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Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.     

Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers

We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication.Antibodies capable of neutralizing a subject''s own virus, called autologous neutralizing antibodies (ANAbs), have been the subject of recent studies redefining the timing and character of this response. ANAbs develop early in essentially all seropositive subjects and increase in titer during the first few months and years of infection (15, 30). Previously published data were obtained using an assay that measures ANAbs against the complete quasispecies without an analysis of the individual envelope protein (Env) sequences to which these ANAb responses were directed (10). The contemporaneous virus pool was poorly neutralized, leading to an assumption that contemporaneous ANAbs are ineffective in controlling viremia. In chronic infection, ANAbs generally have been difficult to detect (3, 29, 31, 40), but there is ample evidence for selection by NAb and resulting virus env evolution in the host (12, 30, 38). The titers of ANAbs measured against clinical or autologous isolates cultured in peripheral blood mononuclear cells typically have been low in chronic infection (31, 40), while other studies indicated the presence of strong ANAbs (2). Although ANAbs may be ineffective in subjects with high virus loads due to the continuous generation of escape variants, their role in maintaining low viral loads in human immunodeficiency virus (HIV) controllers is not known.NAbs that recognize heterologous isolates to which the subject has never been exposed, called heterologous NAbs (HNAbs), are found later in infection, and not all subjects develop this broadening of the response (5). In studies that utilized easy-to-neutralize laboratory or primary viruses, titers of HNAbs can be high (5, 6, 26, 29). Early work had shown that polyclonal HNAbs in HIV-infected subjects are directed to conserved conformational determinants on gp120 (32), including the CD4-binding site (CD4bs) (22). Several human neutralizing monoclonal antibodies with broad activity also are directed to conserved conformational determinants on Env proteins, such as the CD4bs (4) and V3 (17). However, the mechanisms that lead to the development of broad HNAbs are unknown. Their development likely is dependent upon the specific autologous Env proteins to which the subject is exposed, and these proteins are variants of the original infection in these subjects, except for cases of superinfection. Thus, we reasoned that a detailed analysis of the neutralization of individual autologous variants in subjects with broad responses and viral control could be informative.The purpose of this study was to examine the autologous neutralizing responses against autologous viral variants in the plasma of HIV-positive subjects that were controlling infection for many years. These subjects have moderate HNAbs against the quasispecies of other subjects (27). We compared longitudinal samples from five chronically infected, antiretroviral treatment-naive adults late in infection. Despite the short time frame between the sample time points, the amount of env variation was surprisingly high, indicating continuous viral evolution in controllers; contemporaneous ANAbs were present and maintained in all except one elite controller. We cloned individual env gp160 plasma variants and analyzed sequence changes related to the autologous neutralization sensitivity or resistance. We systematically examined the ANAb response directed to individual variants using contemporaneous and noncontemporaneous plasma samples and observed patterns that have not been previously reported. Mutations that were significantly associated with sensitivity or resistance to ANAbs were found on parts of the envelope that are exposed and thus may be accessible to antibodies, consistently with a role in escape and containment by NAbs.     

Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIVmac can coassemble into mature cores of infectious viruses

We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5alpha protein from the rhesus monkey (rhTRIM5alpha) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIV(mac) CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5alpha. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5alpha would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.     

Role of maternal autologous neutralizing antibody in selective perinatal transmission of human immunodeficiency virus type 1 escape variants

Perinatal human immunodeficiency virus type 1 (HIV-1) transmission is characterized by acquisition of a homogeneous viral quasispecies, yet the selective factors responsible for this genetic bottleneck are unclear. We examined the role of maternal autologous neutralizing antibody (aNAB) in selective transmission of HIV-1 escape variants to infants. Maternal sera from 38 infected mothers at the time of delivery were assayed for autologous neutralizing antibody activity against maternal time-of-delivery HIV-1 isolates in vitro. Maternal sera were also tested for cross-neutralization of infected-infant-first-positive-time-point viral isolates. Heteroduplex and DNA sequence analyses were then performed to identify the initial infecting virus as a neutralization-sensitive or escape HIV-1 variant. In utero transmitters (n = 14) were significantly less likely to have aNAB to their own HIV-1 strains at delivery than nontransmitting mothers (n = 17, 14.3% versus 76.5%, P = 0.003). Cross-neutralization assays of infected-infant-first-positive-time-point HIV-1 isolates indicated that while 14/21 HIV-1-infected infant first positive time point isolates were resistant to their own mother's aNAB, no infant isolate was inherently resistant to antibody neutralization by all sera tested. Furthermore, both heteroduplex (n = 21) and phylogenetic (n = 9) analyses showed that selective perinatal transmission and/or outgrowth of maternal autologous neutralization escape HIV-1 variants occurs in utero and intrapartum. These data indicate that maternal autologous neutralizing antibody can exert powerful protective and selective effects in perinatal HIV-1 transmission and therefore has important implications for vaccine development.     

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought.     

Improved vaccine protection from simian AIDS by the addition of nonstructural simian immunodeficiency virus genes

An HIV-1 vaccine able to induce broad CD4+ and CD8+ T cell responses may provide long-term control of viral replication. In this study we directly assess the relative benefit of immunization with vaccines expressing three structural Ags (Gag, Pol, and Env), three early regulatory proteins (Rev, Tat, and Nef), or a complex vaccine expressing all six Ags. The simultaneous administration of all six Ags during vaccination resulted in Ag competition manifested by a relative reduction of CD8+ T cell and lymphoproliferative responses to individual Ags. Despite the Ag competition, vaccination with all six Ags resulted in a delay in the onset and a decrease in the extent of acute viremia after mucosal challenge exposure to highly pathogenic SIV(mac251). Reduced levels of acute viremia correlated with lower post-set point viremia and long-term control of infection. In immunized animals, virus-specific CD4+ T cell and lymphoproliferative responses were preserved during acute viremia, and the maintenance of these responses predicted the long-term virological outcome. Taken together, these results suggest that the breadth of the immune response is probably more important than high frequency responses to a limited number of epitopes. These data provide the first clear evidence of the importance of nonstructural HIV Ags as components of an HIV-1 vaccine.     

Extraepitopic compensatory substitutions partially restore fitness to simian immunodeficiency virus variants that escape from an immunodominant cytotoxic-T-lymphocyte response

Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in gag in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.     

Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine.

Infection of pigtail macaques with SIVsmmPBj14, biological clone 3 (SIV-PBj14-bc13), produces an acute and usually fatal shock-like syndrome 7 to 14 days after infection. We used this simian immunodeficiency virus (SIV) model as a rapid and rigorous challenge to evaluate the efficacy of two SIV Env vaccine strategies. Groups of four pigtail macaques were immunized four times over a 25-week span with either a recombinant Semliki Forest virus expressing the SIV-PBj14 Env gp160 (SFV-SIVgp160) or purified recombinant SIV-PBj14 gp120 (rgp120) in SBN-1 adjuvant. Antibody titers to SIV Env developed in all immunized animals (mean peak titers prior to challenge, 1:1,700 for SFV-SIV gp 160 and 1:10,500 for rgp120), but neither neutralizing antibodies nor SIV-specific T-cell proliferative responses were detectable in any of the vaccinees. All macaques were challenged with a 100% infectious, 75% fatal dose of SIV-PBj14-bc13 at week 26. Three of four control animals died of acute SIV-PBj14 syndrome on days 12 and 13. By contrast, all four SFV-SIVgp160-immunized animals and three of the four rgp120-immunized animals were protected from lethal disease. While all virus-challenged animals became infected, symptoms of the SIV-PBj14 syndrome were more severe in controls than in vaccinees. Mean virus titers in plasma at 13 days postchallenge were approximately 10-fold lower in vaccinated than control animals. However, there was no apparent correlation between survival and levels of peripheral blood mononuclear cell-associated culturable virus, provirus load, or any antiviral immunologic parameter examined. The results indicate that while immunization with SFV-SIVgp160 and rgp120 did not protect against virus infection, these Env vaccines did lower the virus load in plasma and protect against the lethal SIV-PBj14 challenge.     

Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.     

Cellular immunity elicited by human immunodeficiency virus type 1/ simian immunodeficiency virus DNA vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies

High levels of infused anti-human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibodies (MAbs) can completely protect macaque monkeys against mucosal chimeric simian-human immunodeficiency virus (SHIV) infection. Antibody levels below the protective threshold do not prevent infection but can substantially reduce plasma viremia. To assess if HIV-1/SIV-specific cellular immunity could combine with antibodies to produce sterile protection, we studied the effect of a suboptimal infusion of anti-HIV-1 neutralizing antibodies in macaques with active cellular immunity induced by interleukin-2 (IL-2)-adjuvanted DNA immunization. Twenty female macaques were divided into four groups: (i). DNA immunization plus irrelevant antibody, (ii). DNA immunization plus infusion of neutralizing MAbs 2F5 and 2G12, (iii). sham DNA plus 2F5 and 2G12, and (iv). sham DNA plus irrelevant antibody. DNA-immunized monkeys developed CD4 and CD8 T-cell responses as measured by epitope-specific tetramer staining and by pooled peptide ELISPOT assays for gamma interferon-secreting cells. After vaginal challenge, DNA-immunized animals that received irrelevant antibody became SHIV infected but displayed lower plasma viremia than control animals. Complete protection against SHIV challenge occurred in three animals that received sham DNA plus MAbs 2F5 and 2G12 and in two animals that received the DNA vaccine plus MAbs 2F5 and 2G12. Thus, although DNA immunization produced robust HIV-specific T-cell responses, we were unable to demonstrate that these responses contributed to the sterile protection mediated by passive infusion of neutralizing antibodies. These data suggest that although effector T cells can limit viral replication, they are not able to assist humoral immunity to prevent the establishment of initial infection.     

Molecular deconvolution of the neutralizing antibodies induced by an inactivated SARS-CoV-2 virus vaccine

Dear Editor, The rapid emergence and persistence of the pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has had enormous impacts on global health and the economy.Effective vaccines against SARS-CoV-2 are urgently needed to control the coronavirus disease 2019(COVID-19) pandemic,and multiple vaccines have been found to be efficacious in preventing symptomatic COVID-19(Polack et al.,2020;Wu et al.,2020;Jones and Roy,2021).We have developed a traditional beta-propiolactone-inacti-vated aluminum hydroxide-adjuvanted whole-virion SARS-CoV-2 vaccine (BBIBP-CorV),which elicited protective immune responses in clinical trials (Wang et al.,2020;Xia et al.,2021).The vaccine has been granted conditional approvals or emergency use authorizations (EUAs) in China and other countries.     

Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1

Conformationally constrained HIV-1 Env and gp120 immunogens induce broadly cross-reactive neutralizing antibodies. Thus, it is now feasible to rationally design an HIV-1 vaccine that affords protection through humoral mechanisms. This paper reviews our progress toward the development of an oral bacterial vaccine vector that is capable of delivering an HIV-1 DNA vaccine to host lymphoid tissues and inducing broadly neutralizing antibodies to HIV-1 in the mucosal and systemic immune compartments.     

Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.