Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

A clade C HIV-1 vaccine protects against heterologous SHIV infection by modulating IgG glycosylation and T helper response in macaques.

The rising global HIV-1 burden urgently requires vaccines capable of providing heterologous protection. Here, we developed a clade C HIV-1 vaccine consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120) protein, delivered either orally using a needle-free injector or through parenteral injection. We tested protective efficacy of the vaccine against intrarectal challenges with a pathogenic heterologous clade C SHIV infection in rhesus macaques. Both routes of vaccination induced a strong envelope-specific IgG in serum and rectal secretions directed against V1V2 scaffolds from a global panel of viruses with polyfunctional activities. Envelope-specific IgG showed lower fucosylation compared with total IgG at baseline, and most of the vaccine-induced proliferating blood CD4+ T cells did not express CCR5 and α4ß7, markers associated with HIV target cells. After SHIV challenge, both routes of vaccination conferred significant and equivalent protection, with 40% of animals remaining uninfected at the end of six weekly repeated challenges with an estimated efficacy of 68% per exposure. Induction of envelope-specific IgG correlated positively with G1FB glycosylation, and G2S2F glycosylation correlated negatively with protection. Vaccine-induced TNF-α+ IFN-γ+ CD8+ T cells and TNF-α+ CD4+ T cells expressing low levels of CCR5 in the rectum at prechallenge were associated with decreased risk of SHIV acquisition. These results demonstrate that the clade C MVA/CycP-gp120 vaccine provides heterologous protection against a tier2 SHIV rectal challenge by inducing a polyfunctional antibody response with distinct Fc glycosylation profile, as well as cytotoxic CD8 T cell response and CCR5-negative T helper response in the rectum.

Expanding the toolbox of metabolically stable lipid prodrug strategies.

Nucleoside- and nucleotide-based therapeutics are indispensable treatment options for patients suffering from malignant and viral diseases. These agents are most commonly administered to patients as prodrugs to maximize bioavailability and efficacy. While the literature provides a practical prodrug playbook to facilitate the delivery of nucleoside and nucleotide therapeutics, small context-dependent amendments to these popular prodrug strategies can drive dramatic improvements in pharmacokinetic (PK) profiles. Herein we offer a brief overview of current prodrug strategies, as well as a case study involving the fine-tuning of lipid prodrugs of acyclic nucleoside phosphonate tenofovir (TFV), an approved nucleotide HIV reverse transcriptase inhibitor (NtRTI) and the cornerstone of combination antiretroviral therapy (cART). Installation of novel lipid terminal motifs significantly reduced fatty acid hepatic ω-oxidation while maintaining potent antiviral activity. This work contributes important insights to the expanding repertoire of lipid prodrug strategies in general, but particularly for the delivery and distribution of acyclic nucleoside phosphonates.

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion.

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4-6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

T cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers.

Recent efforts toward an HIV vaccine focus on inducing broadly neutralizing antibodies, but eliciting both neutralizing antibodies (nAbs) and cellular responses may be superior. Here, we immunized macaques with an HIV envelope trimer, either alone to induce nAbs, or together with a heterologous viral vector regimen to elicit nAbs and cellular immunity, including CD8+ tissue-resident memory T cells. After ten vaginal challenges with autologous virus, protection was observed in both vaccine groups at 53.3% and 66.7%, respectively. A nAb titer >300 was generally associated with protection but in the heterologous viral vector + nAb group, titers <300 were sufficient. In this group, protection was durable as the animals resisted six more challenges 5 months later. Antigen stimulation of T cells in ex vivo vaginal tissue cultures triggered antiviral responses in myeloid and CD4+ T cells. We propose that cellular immune responses reduce the threshold of nAbs required to confer superior and durable protection.

Vaccine induction of antibodies and tissue-resident CD8+ T cells enhances protection against mucosal SHIV-infection in young macaques.

Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5-expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high-magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.

Adjuvanting a Simian Immunodeficiency Virus Vaccine with Toll-Like Receptor Ligands Encapsulated in Nanoparticles Induces Persistent Antibody Responses and Enhanced Protection in TRIM5α Restrictive Macaques.

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NP-adjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5α restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persistent and protective antibody responses against SIV in RMs with implications for the design of vaccines against human immunodeficiency virus (HIV). IMPORTANCE: The results of the RV144 HIV vaccine trial, which demonstrated a rapid waning of protective immunity with time, have underscored the need to develop strategies to enhance the durability of protective immune responses. Our recent work in mice has highlighted the capacity of nanoparticle-encapsulated TLR ligands (NP) to induce potent and durable antibody responses that last a lifetime in mice. In the present study, we evaluated the ability of these NP adjuvants to promote robust and durable protective immune responses against SIV in nonhuman primates. Our results demonstrate that immunization of rhesus macaques with NP adjuvants mixed with soluble SIV Env or a virus-like particle form of Env (VLP) induces potent and durable Env-specific antibody responses in the serum and in vaginal secretions. These responses were superior to those induced by alum adjuvant, and they resulted in enhanced protection against a low-dose intravaginal challenge with a heterologous strain of SIV in animals with TRIM5a restrictive alleles. These results highlight the potential for such NP TLR L adjuvants in promoting robust and durable antibody responses against HIV in the next generation of HIV immunogens currently being developed.

Effect of Glycosylation on an Immunodominant Region in the V1V2 Variable Domain of the HIV-1 Envelope gp120 Protein.

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchange molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. These studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to focus the immune response on key neutralizing epitopes.

Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2.

Signatures in Simian Immunodeficiency Virus SIVsmE660 Envelope gp120 Are Associated with Mucosal Transmission but Not Vaccination Breakthrough in Rhesus Macaques.

UNLABELLED: Mucosal surfaces are vulnerable to human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection and thus are key sites for eliciting vaccine-mediated protection. Vaccine protocols carried out at the Yerkes Primate Research Center utilized SIVmac239-based immunization strategies with intrarectal and intravaginal SIVsmE660 challenge of rhesus macaques. We investigated whether there were genetic signatures associated with SIVsmE660 intrarectal and intravaginal transmissions in vaccinated and unvaccinated monkeys. When transmitted/founder (T/F) envelope (Env) sequences from 49 vaccinated and 15 unvaccinated macaques were compared to each other, we were unable to identify any vaccine breakthrough signatures. In contrast, when the vaccinated and control T/F Envs were combined and compared to the challenge stock, residues at gp120 positions 23, 45, 47, and 70 (Ile-Ala-Lys-Asn [I-A-K-N]) emerged as signatures of mucosal transmission. However, T/F Envs derived from intrarectal and intravaginal infections were not different. Our data suggest that the vaginal and rectal mucosal environments both imposed a strong selection bias for SIVsmE660 variants carrying I-A-K-N that was not further enhanced by immunization. These findings, combined with the strong conservation of A-K-N in most HIV-2/SIVsmm isolates and the analogous residues in HIV-1/SIVcpz isolates, suggest that these residues confer increased transmission fitness to SIVsmE660. IMPORTANCE: Most HIV-1 infections occur across a mucosal barrier, and it is therefore important to understand why these sites are vulnerable and how to protect them with a vaccine. To gain insight into these questions, we studied rhesus macaques that were vaccinated with SIVmac239 and unvaccinated controls to determine whether the SIVsmE660 viral variants that infected these two groups were different. We did not find differences between viral variants in the absence versus presence of vaccination-induced immunity, but we did find that the SIVsmE660 viral variants that infected the monkeys, regardless of vaccination, were different from the dominant population found in the viral challenge inoculum. Our data suggest that the mucosal environments of the vagina and rectum both impose a strong selection for the SIVsmE660 variants in the challenge inoculum that are most like SIV and HIVs that circulate in nature.

Breakthrough of SIV strain smE660 challenge in SIV strain mac239-vaccinated rhesus macaques despite potent autologous neutralizing antibody responses.

Although the correlates of immunological protection from human immunodeficiency virus or simian immunodeficiency virus infection remain incompletely understood, it is generally believed that medium to high titers of serum neutralizing antibodies (nAbs) against the challenge virus will prevent infection. This paradigm is based on a series of studies in which passive transfer of HIV-specific nAbs protected rhesus macaques (RMs) from subsequent mucosal challenge with a chimeric human/simian immunodeficiency virus. However, it is unknown whether nAb titers define protection in the setting of active immunization. Here we determined serum nAb titers against breakthrough transmitted/founder (T/F) SIVsmE660-derived envelope glycoprotein (Env) variants from 14 RMs immunized with SIVmac239-based DNA-prime/modified vaccinia virus Ankara-boost vaccine regimens that included GM-CSF or CD40L adjuvants and conferred significant but incomplete protection against repeated low-dose intrarectal challenge. A single Env variant established infection in all RMs except one, with no identifiable genetic signature associated with vaccination breakthrough compared with T/F Envs from four unvaccinated monkeys. Breakthrough T/F Env pseudoviruses were potently neutralized in vitro by heterologous pooled serum from chronically SIVsmE660-infected monkeys at IC50 titers exceeding 1:1,000,000. Remarkably, the T/F Env pseudoviruses from 13 of 14 monkeys were also susceptible to neutralization by autologous prechallenge serum at in vitro IC50 titers ranging from 1:742-1:10,832. These titers were similar to those observed in vaccinated RMs that remained uninfected. These data suggest that the relationship between serum nAb titers and protection from mucosal SIV challenge in the setting of active immunization is more complex than previously recognized, warranting further studies into the balance between immune activation, target cell availability, and protective antibody responses.

Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope.

UNLABELLED: Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen. IMPORTANCE: Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

HIV-1 non-macrophage-tropic R5 envelope glycoproteins are not more tropic for entry into primary CD4+ T-cells than envelopes highly adapted for macrophages.

BACKGROUND: Non-mac-tropic HIV-1 R5 viruses are predominantly transmitted and persist in immune tissue even in AIDS patients who carry highly mac-tropic variants in the brain. Non-mac-tropic R5 envelopes (Envs) require high CD4 levels for infection contrasting with highly mac-tropic Envs, which interact more efficiently with CD4 and mediate infection of macrophages that express low CD4. Non-mac-tropic R5 Envs predominantly target T-cells during transmission and in immune tissue where they must outcompete mac-tropic variants. Here, we investigated whether Env+ pseudoviruses bearing transmitted/founder (T/F), early and late disease non-mac-tropic R5 envelopes mediated more efficient infection of CD4+ T-cells compared to those with highly mac-tropic Envs. RESULTS: Highly mac-tropic Envs mediated highest infectivity for primary T-cells, Jurkat/CCR5 cells, myeloid dendritic cells, macrophages, and HeLa TZM-bl cells, although this was most dramatic on macrophages. Infection of primary T-cells mediated by all Envs was low. However, infection of T-cells was greatly enhanced by increasing virus attachment with DEAE dextran and spinoculation, which enhanced the three Env+ virus groups to similar extents. Dendritic cell capture of viruses and trans-infection also greatly enhanced infection of primary T-cells. In trans-infection assays, non-mac-tropic R5 Envs were preferentially enhanced and those from late disease mediated levels of T-cell infection that were equivalent to those mediated by mac-tropic Envs. CONCLUSIONS: Our results demonstrate that T/F, early or late disease non-mac-tropic R5 Envs do not preferentially mediate infection of primary CD4+ T-cells compared to highly mac-tropic Envs from brain tissue. We conclude that non-macrophage-tropism of HIV-1 R5 Envs in vitro is determined predominantly by a reduced capacity to target myeloid cells via low CD4 rather than a specific adaptation for T-cells entry that precludes macrophage infection.

CD4 depletion in SIV-infected macaques results in macrophage and microglia infection with rapid turnover of infected cells.

In rhesus macaques (RMs), experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

Decreased plasticity of coreceptor use by CD4-independent SIV Envs that emerge in vivo.

BACKGROUND: HIV and SIV generally require CD4 binding prior to coreceptor engagement, but Env can acquire the ability to use CCR5 independently of CD4 under various circumstances. The ability to use CCR5 coupled with low-to-absent CD4 levels is associated with enhanced macrophage infection and increased neutralization sensitivity, but the additional features of these Envs that may affect cell targeting is not known. RESULTS: Here we report that CD4-independent SIV variants that emerged in vivo in a CD4+ T cell-depleted rhesus macaque model display markedly decreased plasticity of co-receptor use. While CD4-dependent Envs can use low levels of macaque CCR5 for efficient entry, CD4-independent variants required high levels of CCR5 even in the presence of CD4. CD4-independent Envs were also more sensitive to the CCR5 antagonist Maraviroc. CD4-dependent variants mediated efficient entry using human CCR5, whereas CD4-independent variants had impaired use of human CCR5. Similarly, CD4-independent Envs used the alternative coreceptors GPR15 and CXCR6 less efficiently than CD4-dependent variants. Env amino acids D470N and E84K that confer the CD4-independent phenotype also regulated entry through low CCR5 levels and GPR15, indicating a common structural basis. Treatment of CD4-dependent Envs with soluble CD4 enhanced entry through CCR5 but reduced entry through GPR15, suggesting that induction of CD4-induced conformational changes by non-cell surface-associated CD4 impairs use of this alternative co-receptor. CONCLUSIONS: CD4 independence is associated with more restricted coreceptor interactions. While the ability to enter target cells through CCR5 independently of CD4 may enable infection of CD4 low-to-negative cells such as macrophages, this phenotype may conversely reduce the potential range of targets such as cells expressing low levels of CCR5, conformational variants of CCR5, or possibly even alternative coreceptors.

CD4+ T cells support production of simian immunodeficiency virus Env antibodies that enforce CD4-dependent entry and shape tropism in vivo.

CD4(+) T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4(+) T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4(+) T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV(+)) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV(+) plasma neutralization. However, plasma from SIV-infected CD4(+) T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4(+) T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4(+) target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.

Subtype-specific conservation of isoleucine 309 in the envelope V3 domain is linked to immune evasion in subtype C HIV-1 infection.

The V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key functional domain yet it exhibits distinct mutational patterns across subtypes. Here an invariant residue (Ile 309) was replaced with Leu in 7 subtype C patient-derived Envs from recent infection and 4 related neutralizing antibody escape variants that emerged later. For these 11 Envs, I309L did not alter replication in primary CD4 T cells; however, replication in monocyte-derived macrophages was enhanced. Infection of cell lines with low CD4 or CCR5 revealed that I309L enhanced utilization of CD4 but did not affect the ability to use CCR5. This CD4-enhanced phenotype tracked with sensitivity to sCD4, indicating increased exposure of the CD4 binding site. The results suggest that Ile 309 preserves a V3-mediated masking function that occludes the CD4 binding site. The findings point to an immune evasion strategy in subtype C Env to protect this vulnerable immune target.

Heterosexual transmission of human immunodeficiency virus type 1 subtype C: Macrophage tropism, alternative coreceptor use, and the molecular anatomy of CCR5 utilization.

Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.

Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection.

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4(+) T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12-20 mo, viruses exhibited concentrated mutations at 17-34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes.

Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor-operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This system therefore could facilitate the development of Ad-based vaccine candidates that otherwise would not be feasible to generate.