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The explosion of SARS-CoV-2 infections in 2020 prompted a flurry of activity in vaccine development and exploration of various vaccine platforms, some well-established and some new. Phage-based vaccines were described previously, and we explored the possibility of using mycobacteriophages as a platform for displaying antigens of SARS-CoV-2 or other infectious agents. The potential advantages of using mycobacteriophages are that a large and diverse variety of them have been described and genomically characterized, engineering tools are available, and there is the capacity to display up to 700 antigen copies on a single particle approximately 100 nm in size. The phage body may itself be a good adjuvant, and the phages can be propagated easily, cheaply, and to high purity. Furthermore, the recent use of these phages therapeutically, including by intravenous administration, suggests an excellent safety profile, although efficacy can be restricted by neutralizing antibodies. We describe here the potent immunogenicity of mycobacteriophage Bxb1, and Bxb1 recombinants displaying SARS-CoV-2 Spike protein antigens.

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences., Author summary Use of an entry coreceptor, in conjunction with CD4, is the main determinant of HIV/SIV cell targeting, which in turn may be a central factor determining pathogenicity of infection. Two SIVs that generally do not cause AIDS in their natural hosts, SIVsmm of sooty mangabeys and SIVagm of African green monkeys, express low levels of the canonical coreceptor CCR5 and have been shown recently to enter CD4+ T cells via CXCR6 in addition to CCR5. Here we asked which entry coreceptors are used by SIVcpz, the HIV-1 precursor that is pathogenic in chimpanzees, and by SIVmus, a member of the monkey SIV lineage that contributed the env gene of SIVcpz. We found that SIVcpz uses only CCR5, while SIVmus efficiently uses both CXCR6 and CCR5 for entry. We then examined CXCR6 expression on sooty mangabey CD4+ T cells, and found it is present on a subset of differentiated memory T cells distinct from cells expressing CCR5. Coreceptor-mediated cell targeting thus differs among primate lentiviruses. Entry via CXCR6 in some primate hosts may target SIV towards cells that may support viremia without causing immunodeficiency, but this pattern was lost during the emergence of pathogenic SIVcpz and HIV-1 infections.

Current approaches do not eliminate all HIV-1 maternal-to-infant transmissions (MTIT); new prevention paradigms might help avert new infections. We administered Maraviroc (MVC) to rhesus macaques (RMs) to block CCR5-mediated entry, followed by repeated oral exposure of a CCR5-dependent clone of simian immunodeficiency virus (SIV)mac251 (SIVmac766). MVC significantly blocked the CCR5 coreceptor in peripheral blood mononuclear cells and tissue cells. All control animals and 60% of MVC-treated infant RMs became infected by the 6th challenge, with no significant difference between the number of exposures (p=0.15). At the time of viral exposures, MVC plasma and tissue (including tonsil) concentrations were within the range seen in humans receiving MVC as a therapeutic. Both treated and control RMs were infected with only a single transmitted/founder variant, consistent with the dose of virus typical of HIV-1 infection. The uninfected RMs expressed the lowest levels of CCR5 on the CD4+ T cells. Ramp-up viremia was significantly delayed (p=0.05) in the MVC-treated RMs, yet peak and postpeak viral loads were similar in treated and control RMs. In conclusion, in spite of apparent effective CCR5 blockade in infant RMs, MVC had marginal impact on acquisition and only a minimal impact on post infection delay of viremia following oral SIV infection. Newly developed, more effective CCR5 blockers may have a more dramatic impact on oral SIV transmission than MVC.ImportanceWe have previously suggested that the very low levels of simian immunodeficiency virus (SIV) maternal-to-infant transmissions (MTIT) in African nonhuman primates that are natural hosts of SIVs are due to a low availability of target cells (CCR5+ CD4+ T cells) in the oral mucosa of the infants, rather than maternal and milk factors. To confirm this new MTIT paradigm, we performed a proof of concept study, in which we therapeutically blocked CCR5 with maraviroc (MVC) and orally exposed MVC treated and naïve infant rhesus macaques to SIV. MVC had only a marginal effect on oral SIV transmission. However, the observation that the infant RMs that remained uninfected at the completion of the study, after 6 repeated viral challenges, had the lowest CCR5 expression on the CD4+ T cells prior to the MVC treatment, appear to confirm our hypothesis, also suggesting that the partial effect of MVC is due to a limited efficacy of the drug. Newly, more effective CCR5 inhibitors may have a better effect in preventing SIV and HIV transmission.

Natural-host sooty mangabeys (SM) infected with simian immunodeficiency virus (SIV) exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4 + T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which have been linked to low expression of the canonical SIV entry receptor CCR5 on CD4 + T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by SIV in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIV smm in vitro . Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIV infection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4 + T cell subset targeting compared with that in nonnatural hosts, enabling SIV to maintain high-level replication without leading to widespread CD4 + T cell loss. IMPORTANCE Natural hosts of SIV, such as sooty mangabeys, sustain high viral loads but do not develop disease, while nonnatural hosts, like rhesus macaques, develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5, suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may redirect the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of nonessential cells and limit infection of critical cells in natural hosts, thus contributing to benign outcome of infection.

Pathogenic HIV-1 infection of humans and SIVmac infection of macaques are the result of zoonotic transfer of primate immunodeficiency viruses from their natural hosts into non-natural host species. Natural host infections do not result in pathogenesis despite high levels of virus replication, and evidence suggests that differences in anatomical location and specific subsets of CD4+ T cells infected may underlie distinct outcomes from infection. The coreceptor CCR5 has long been considered the sole pathway for SIV entry and the key determinant of CD4+ cell targeting, but it has also been known that natural hosts express exceedingly low levels of CCR5 despite maintaining high levels of virus replication. This review details emerging data indicating that in multiple natural host species, CCR5 is dispensable for SIV infection ex vivo and/or in vivo and, contrary to the established dogma, alternative coreceptors, particularly CXCR6, play a central role in infection and cell targeting. Infections of non-natural hosts, however, are characterized by CCR5-exclusive entry. These findings suggest that alternative coreceptor-mediated cell targeting in natural hosts, combined with low CCR5 expression, may direct the virus to distinct populations of cells that are dispensable for immune homeostasis, particularly extralymphoid and more differentiated CD4+ T cells. In contrast, CCR5-mediated entry in non-natural hosts results in targeting of CD4+ T cells that are located in lymphoid tissues, critical for immune homeostasis, or necessary for gut barrier integrity. Thus, fundamental differences in viral entry coreceptor use may be central determinants of infection outcome. These findings redefine the normal SIV/host relationship in natural host species, shed new light on key features linked to zoonotic immunodeficiency virus transfer, and highlight important questions regarding how and why this coreceptor bottleneck occurs and the coevolutionary equilibrium is lost following cross-species transfer that results in AIDS.

Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites.

African green monkeys (AGM) and sooty mangabeys (SM) are well-studied natural hosts of simian immunodeficiency virus (SIV) that do not progress to AIDS when infected with their species-specific viruses. Natural hosts of SIV express very low levels of the canonical entry coreceptor CCR5, and recent studies have shown that CCR5 is dispensable for SIV infection of SM in vivo and that blocking of CCR5 does not prevent ex vivo infection of peripheral blood mononuclear cells (PBMC) from SM or vervet AGM. In both hosts, CXCR6 is an efficient entry pathway in vitro . Here we investigated the use of species-matched CXCR6 and other alternative coreceptors by SIVagmSab, which infects sabaeus AGM. We cloned sabaeus CD4 and 10 candidate coreceptors. Species-matched CXCR6, CCR5, and GPR15 mediated robust entry into transfected cells by pseudotypes carrying SIVagmSab92018ivTF Env, with lower-level entry through GPR1 and APJ. We cloned genetically divergent env genes from the plasma of two wild-infected sabaeus AGM and found similar patterns of coreceptor use. Titration experiments showed that CXCR6 and CCR5 were more efficient than other coreceptors when tested at limiting CD4/coreceptor levels. Finally, blocking of CXCR6 with its ligand CXCL16 significantly inhibited SIVagmSab replication in sabaeus PBMC and had a greater impact than did the CCR5 blocker maraviroc, confirming the use of CXCR6 in primary lymphocyte infection. These data suggest a new paradigm for SIV infection of natural host species, whereby a shared outcome of virus-host coevolution is the use of CXCR6 or other alternative coreceptors for entry, which may direct SIV toward CD4 + T cell subsets and anatomical sites that support viral replication without disrupting immune homeostasis and function. IMPORTANCE Natural hosts of SIV do not progress to AIDS, in stark contrast to pathogenic human immunodeficiency virus type 1 (HIV-1)-human and SIVmac-macaque infections. Identifying how natural hosts avoid immunodeficiency can elucidate key mechanisms of pathogenesis. It is known that despite high viral loads, natural hosts have a low frequency of CD4 + cells expressing the SIV coreceptor CCR5. In this study, we demonstrate the efficient use of the coreceptor CXCR6 by SIVagmSab to infect sabaeus African green monkey lymphocytes. In conjunction with studies of SIVsmm, which infects sooty mangabeys, and SIVagmVer, which infects vervet monkeys, our data suggest a unifying model whereby in natural hosts, in which the CCR5 expression level is low, the use of CXCR6 or other coreceptors to mediate infection may target SIV toward distinct cell populations that are able to support high-level viral replication without causing a loss of CD4 + T cell homeostasis and lymphoid tissue damage that lead to AIDS in HIV-1 and SIVmac infections.

Human immunodeficiency virus, a primate lentivirus (PLV), causes AIDS in humans, whereas most PLVs are less or not pathogenic in monkeys. These notions suggest that the co-evolutionary process of PLVs and their hosts associates with viral pathogenicity and therefore, that elucidating the history of virus-host co-evolution is one of the most intriguing topics in the field of virology. To address this, recent studies have focused on the interplay between intrinsic anti-viral proteins, such as tetherin and viral antagonists. Through an experimental-phylogenetic approach, here we investigate the co-evolutionary interplay between tribe Cercopithecini tetherin and viral antagonists, Nef and Vpu. We reveal that tribe Cercopithecini tetherins are positively selected, possibly triggered by ancient Nef-like factor(s). We reconstruct the ancestral sequence of tribe Cercopithecini tetherin and demonstrate that all Nef proteins are capable of antagonizing ancestral Cercopithecini tetherin. Further, we consider the significance of evolutionary arms race between tribe Cercopithecini and their PLVs.

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.

An effective T-cell-based AIDS vaccine should induce strong HIV-specific CD8(+) T cells in mucosal tissues without increasing the availability of target cells for the virus. Here, we evaluated five immunization strategies that include Human adenovirus-5 (AdHu5), Chimpanzee adenovirus-6 (AdC6) or -7 (AdC7), Vaccinia virus (VV), and DNA given by electroporation (DNA/EP), all expressing Simian immunodeficiency virus group specific antigen/transactivator of transcription (SIV(mac239Gag/Tat)). Five groups of six rhesus macaques (RMs) each were vaccinated with DNA/EP-AdC6-AdC7, VV-AdC6-AdC7, DNA/-EP-VV-AdC6, DNA/EP-VV-AdC7, or AdHu5-AdHu5-AdHu5 and were challenged repeatedly with low-dose intrarectal SIVmac239. Upon challenge, there were no significant differences among study groups in terms of virus acquisition or viral load after infection. When taken together, the immunization regimens did not protect against SIV acquisition compared with controls but did result in an ∼ 1.6-log decline in set-point viremia. Although all immunized RMs had detectable SIV-specific CD8(+) T cells in blood and rectal mucosa, we found no correlation between the number or function of these SIV-specific CD8(+) T cells and protection against SIV acquisition. Interestingly, RMs experiencing breakthrough infection showed significantly higher prechallenge levels of CD4(+)C-C chemokine receptor type 5 (CCR5)(+)HLA-DR(+) T cells in the rectal biopsies (RB) than animals that remained uninfected. In addition, among the infected RMs, the percentage of CD4(+)CCR5(+)Ki-67(+) T cells in RBs prechallenge correlated with higher early viremia. Overall, these data suggest that the levels of activated CD4(+)CCR5(+) target T cells in the rectal mucosa may predict the risk of SIV acquisition in RMs vaccinated with vectors that express SIVGag/Tat.