Is the Gut the Major Source of Virus in Early Simian Immunodeficiency Virus Infection?

The acute phases of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection are characterized by rapid and profound depletion of CD4⁺ T cells from the guts of infected individuals. The large number of CD4⁺ T cells in the gut (a large fraction of which are activated and express the HIV/SIV coreceptor CCR5), the high level of infection of these cells, and the temporal coincidence of this CD4⁺ T-cell depletion with the peak of virus in plasma in acute infection suggest that the intestinal mucosa may be the major source of virus driving the peak viral load. Here, we used data on CD4⁺ T-cell proportions in the lamina propria of the rectums of SIV-infected rhesus macaques (which progress to AIDS) and sooty mangabeys (which do not progress) to show that in both species, the depletion of CD4⁺ T cells from this mucosal site and its maximum loss rate are often observed several days before the peak in viral load, with few CD4⁺ T cells remaining in the rectum by the time of peak viral load. In contrast, the maximum loss rate of CD4⁺ T cells from bronchoalveolar lavage specimens and lymph nodes coincides with the peak in virus. Analysis of the kinetics of depletion suggests that, in both rhesus macaques and sooty mangabeys, CD4⁺ T cells in the intestinal mucosa are a highly susceptible population for infection but not a major source of plasma virus in acute SIV infection.The acute phase of human immunodeficiency virus (HIV) infection is characterized by moderate CD4⁺ T-cell depletion in blood, followed by a transient partial restoration of CD4⁺ T-cell numbers and eventually by a slow long-term CD4⁺ T-cell decline in the chronic phase that lasts for several years. Studies of CD4⁺ T-cell depletion in mucosal sites, often conducted with simian immunodeficiency virus (SIV)-infected macaques, have demonstrated that mucosal CD4⁺ T-cell depletion is more rapid and profound (3, 10, 13, 19, 21). The severe depletion of cells in the gut in early infection is thought to be driven in part by the phenotype of the cells present, which are predominantly CCR5⁺ and in general more activated than their circulating counterparts. As such, these mucosal CD4⁺ T cells are highly susceptible to productive infection with the dominant CCR5-tropic strains of HIV and SIV present in early infection (20). The rapid depletion of CD4⁺ T cells at mucosal sites is accompanied by relatively high numbers of infected cells (10, 13) and is temporally associated with the peak viral load in plasma, suggesting that the infection of mucosal CD4⁺ T cells may be responsible for the majority of virus replication occurring during acute infection (10, 15, 21, 22).The size of the CD4⁺ T-cell pool in the gut is a matter of some controversy, with estimates ranging from ∼5 to 50% of the total body pool of these cells (reviewed in reference 5). Regardless of the precise numbers, the gut (and particularly the mucosal lamina propria) contains a significant proportion of the body CD4⁺ CCR5⁺ memory T cells, which are depleted very early in infection. However, whether CD4⁺ T cells in the gut are merely a target of early infection or whether they are a major driver of early viral growth and peak viral loads in acute infection is unclear. Here we use a combination of experimental data and modeling to demonstrate that the gut is unlikely to be a major source of virus production in acute SIV infection.     

Simian Virus 40 Mutants with Deletions at the 3′ End of the Early Region Are Defective in Adenovirus Helper Function

Coinfection of monkey cells with simian virus 40 (SV40) and adenovirus type 2 (Ad2) increased the Ad2 yield 1,000-fold over that obtained by Ad2 infection alone of monkey cells (A. S. Rabson, G. T. O'Conor, I. K. Berezesky, and F. J. Paul, Proc. Soc. Exp. Biol. Med. 116:187-190, 1964). The ability of viable mutants of SV40 that contain deletions at various sites in the viral DNA to enhance Ad2 growth in monkey cells was examined. Only those mutants with deletions near the 3' end of the early region were deficient in providing this helper function. Mutants dl1265, lacking 39 base pairs at map position 0.18, and dl1263, lacking 33 base pairs at map position 0.20 (H. van Heuverswyn, C. Cole, P. Berg, and W. Fiers, J. Virol. 30:936-941, 1979), were approximately 4 and 30% as effective as wild-type SV40, respectively. The extent of enhancement of Ad2 yield depended on the multiplicity of infection by SV40, but not by Ad2 (at a multiplicity of infection of 相似文献   

Simian Virus 40 Activates ATR-Δp53 Signaling To Override Cell Cycle and DNA Replication Control

During infection, simian virus 40 (SV40) attempts to take hold of the cell, while the host responds with various defense systems, including the ataxia-telangiectasia mutated/ATM-Rad3 related (ATM/ATR)-mediated DNA damage response pathways. Here we show that upon viral infection, ATR directly activates the p53 isoform Δp53, leading to upregulation of the Cdk inhibitor p21 and downregulation of cyclin A-Cdk2/1 (AK) activity, which force the host to stay in the replicative S phase. Moreover, downregulation of AK activity is a prerequisite for the generation of hypophosphorylated, origin-competent DNA polymerase α-primase (hypo-Polα), which is, unlike AK-phosphorylated Polα (P-Polα), recruited by SV40 large T antigen (T-Ag) to initiate viral DNA replication. Prevention of the downregulation of AK activity by inactivation of ATR-Δp53-p21 signaling significantly reduced the T-Ag-interacting hypo-Polα population and, accordingly, SV40 replication efficiency. Moreover, the ATR-Δp53 pathway facilitates the proteasomal degradation of the 180-kDa catalytic subunit of the non-T-Ag-interacting P-Polα, giving rise to T-Ag-interacting hypo-Polα. Thus, the purpose of activating the ATR-Δp53-p21-mediated intra-S checkpoint is to maintain the host in S phase, an optimal environment for SV40 replication, and to modulate the host DNA replicase, which is indispensable for viral amplification.Infection of quiescent CV-1 cells with the primate polyomavirus simian virus 40 (SV40) induces cell cycle progression and stimulates host cell DNA replication, which is mandatory for viral amplification. SV40 uses only a single viral protein, T antigen (T-Ag), for its own replication; all other components have to be provided by the host. Initially, a specifically phosphorylated subclass of T-Ag binds to a palindromic sequence in the SV40 origin (43), and in the presence of ATP, T-Ag forms a double-hexamer nucleoprotein complex leading to structural distortion and unwinding of origin DNA sequences (5). In concert with the cellular single-strand DNA binding protein RPA and topoisomerase I, the DNA helicase activity of T-Ag promotes more-extensive origin unwinding, forming a preinitiation complex (pre-RC), resulting in an initiation complex (53). Once the initiation complex forms, the primase activity of the heterotetrameric DNA polymerase α-primase (Polα) complex, consisting of the p180 catalytic subunit, the p70 regulatory subunit, and the p48/58 primase subunits, synthesizes a short RNA primer on each template strand, which is extended by the DNA polymerase activity of Polα (6, 17). Immediately after the first nascent RNA/DNA primer is synthesized, the complete replication machinery is assembled, and elongation at both forks by the processive DNA polymerase δ ensues (62). Thus, during the initiation of SV40 replication, T-Ag performs many of the functions attributed to the eukaryotic pre-RC complex proteins, including Orc, Cdc6, Cdt1, and kinase-independent cyclin E, which facilitates loading of the putative replication helicase Mcm2-7 onto the eukaryotic origin (4, 18). Biochemical evidence shows that initiation of SV40 and eukaryotic DNA replication occurs by the physical interaction of Polα with the appropriate pre-RC in the immediate vicinity of the origin. In SV40, Polα is loaded onto the origin by direct physical contact between the helicase T-Ag and its p180 N-terminal domain C (14, 15, 16). In eukaryotes, Cdc45, Mcm10, and And-1 cooperate to recruit Polα to the origin-initiation complex, thereby tethering the replicase to the origin-loaded Mcm2-7 helicase (34, 61).Although SV40 and chromosomal DNA replication share the same essential replication factors that are recruited to the appropriate pre-RC, there are noticeable differences between the SV40 and eukaryotic replication systems. The viral system allows unregulated multiple firing of the origin, whereas in the eukaryotic system, origin-dependent initiation of replication is regulated and restricted to firing only once per cell cycle. Reinitiation at origins within a cell cycle is prevented by the inactivation of pre-RC components in S and G₂. The cyclin-dependent kinases (Cdks) play a central role in establishing a block to rereplication through phosphorylation of each of the components. At present, several proteins of the mammalian pre-RC, such as Orc1, Cdt1, Cdc6, and the Mcm complex are phosphorylated by cyclin A (cycA)-Cdk2/1 (AK) and, as a result, are degraded or inactivated (1, 26, 30, 33, 40). Nevertheless, not all of the pre-RC components mentioned above are utilized by SV40, and accordingly, not all are involved in viral initiation control. However, in both replication systems, DNA synthesis is initiated by Polα and its initiation activity is regulated by Cdks (55). Moreover, AK-phosphorylated Polα is not recruited to mammalian origins in vivo (13) and is unable to initiate SV40 replication in vitro (47, 57, 58). Considering that cellular mechanisms blocking the rereplication of DNA act by AK phosphorylation of the replication factors mentioned above, especially Polα, it is feasible to suggest that downregulation or even inhibition of this kinase activity promotes dysregulation of replication control in SV40-infected cells.One pathway that leads to downregulation of AK activity in response to cellular stress is the intra-S checkpoint, which employs the novel p53 isoform Δp53 (45). In UV-damaged S-phase cells, ATR (ataxia-telangiectasia mutated [ATM]-Rad3 related)-activated Δp53 upregulates the Cdk inhibitor p21, resulting in a transient reduction in AK activity and decelerated S-phase progression (45). Here we demonstrate that SV40 lytic infection triggers the ATR signaling cascade, leading to the activation of Δp53 and accordingly a p21-mediated drop in AK activity to prevent AK-catalyzed inactivation of the hypophosphorylated, T-Ag-interacting Polα (hypo-Polα) subclass, which is essential for the initiation of viral DNA replication.     

Infection with “Escaped” Virus Variants Impairs Control of Simian Immunodeficiency Virus SIVmac239 Replication in Mamu-B*08-Positive Macaques

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08⁺) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8⁺ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8⁺ T-cell epitopes to investigate the contribution of epitope-specific CD8⁺ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08⁺ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08⁺ macaques. The five most immunodominant Mamu-B*08-restricted CD8⁺ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08⁺ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08⁺ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8⁺ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08⁺ macaques.A few individuals spontaneously control the replication of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) to very low levels. The precise mechanisms underlying this control are of great interest, as a clear understanding of what constitutes a successful immune response may aid in developing an AIDS vaccine. Particularly pressing questions for vaccine design include which proteins to use as immunogens, the extent to which increasing the breadth and magnitude of responses is advantageous, how immunodomination affects T-cell responses, and if biasing the immune response toward particular effector profiles is beneficial. Characterization of immune responses made by elite controllers (ECs) may reveal patterns that can then be applied to vaccine formulation and evaluation.HIV ECs are generally not infected with grossly unfit viruses (6, 42). Instead, elite control of immunodeficiency virus replication is correlated with the presence of particular major histocompatibility complex class I (MHC-I) alleles (11, 12, 18, 32, 41, 55). The association of MHC-I alleles with the control of viremia implicates CD8⁺ T cells as being mediators of this immune containment. Several lines of evidence support this hypothesis. These lines of evidence include the correlation between the appearance of CD8⁺ T-cell responses and the resolution of peak viremia during acute infection (7, 29), the finding that alleles associated with viral control restrict dominant acute-phase CD8⁺ T-cell responses (3), and the finding that responses directed against epitopes restricted by these alleles frequently select for viral escape variants (4, 27, 38). Perhaps most compelling is the observation that for a few HIV-infected individuals, the selection of escape variants by an immunodominant HLA-B27-restricted T-cell response temporally preceded substantial increases in viremia (17, 21, 53). While viruses exhibiting escape variants in epitopes restricted by protective alleles are often detectably less fit in vitro (10, 38, 43, 51), recent data have found normal, high levels of replication in vivo upon the transmission of some of these variants (15).The association of control with MHC-I alleles does not, of course, implicate solely CD8⁺ T cells. MHC-I molecules are also ligands for killer immunoglobulin receptors (KIRs), which are predominantly expressed on natural killer (NK) cells. Genetic studies of HIV-infected humans suggest a model in which individuals with particular KIR/HLA combinations are predisposed to control HIV replication more readily than those with other KIR/HLA combinations (36, 37). These data were supported by functional studies of this KIR/HLA pairing in vitro, which demonstrated an inhibition of HIV replication by such NK cells (2). The relative contributions of NK and CD8⁺ T-cell responses to control have yet to be elucidated and may be closely intertwined.Previously, the experimental depletion of circulating CD8⁺ cells from SIVmac239-infected ECs resulted in a sharp spike in viremia, which resolved as CD8⁺ cells repopulated the periphery (19). During the reestablishment of control of SIV replication, CD8⁺ T cells targeting multiple epitopes restricted by alleles associated with elite control expanded in frequency, providing strong circumstantial evidence for their role in maintaining elite control (19, 31). However, CD8 depletion antibodies used in macaques also remove NK cells, which, at least in vitro, also inhibit SIV replication (19). It was therefore difficult to make definitive conclusions regarding the separate contributions of these subsets to maintaining the control of SIV replication in vivo.Here we investigate elite control in the rhesus macaque model for AIDS. We focused on the macaque MHC-I allele most tightly associated with the control of SIVmac239, Mamu-B*08. Approximately 50% of Mamu-B*08-positive (Mamu-B*08⁺) animals infected with SIVmac239 become ECs (32). Peptides presented by Mamu-B*08 share a binding motif with peptides presented by HLA-B27. Although these two MHC-I genes are dissimilar in domains that are important for peptide binding, each molecule can bind peptides that are presented by the other molecule (33). This striking similarity suggests that the elite control of SIVmac239 in Mamu-B*08⁺ animals is a good model for the elite control of HIV.Seven SIVmac239 epitopes restricted by Mamu-B*08 accrue variation in Mamu-B*08⁺ rhesus macaques (30, 31). For an eighth Mamu-B*08-restricted epitope, which is also restricted by Mamu-B*03 (Mamu-B*03 differs from Mamu-B*08 by 2 amino acids in the α1 and α2 domains [9, 32]), escape has been documented only for SIV-infected Mamu-B*03⁺ macaques (16). Variation in these CD8⁺ T-cell epitopes accumulates with different kinetics, starting during acute infection for those targeted by high-magnitude responses.In this study, we addressed the question of whether the elite control of SIVmac239 in Mamu-B*08⁺ animals is mediated by the known high-frequency CD8⁺ T-cell responses targeting Mamu-B*08-restricted epitopes. To this end, we introduced point mutations into eight epitopes, with the goal of reducing or abrogating immune responses directed against these epitopes during acute infection. We hypothesized that Mamu-B*08⁺ macaques would be unable to control SIV replication without these Mamu-B*08-restricted T-cell responses.     

Temperature-Sensitive Simian Virus 40-Transformed Cells: Phenomena Accompanying Transition from the Transformed to the “Normal” State

Temperature-sensitive simian virus (SV 40)-transformed 3T3 cells (tsSV3T3), which express the transformed phenotype when growing at 32 C but not at 39 C, were used to study changes in growth behavior during shift-up or shift-down experiments. In cultures of tsSV3T3 cells which had reached or were beyond monolayer density at 32 C, DNA synthesis reached very low levels within 24 to 48 h after shift-up. When cells which had been allowed to grow to high densities at 32 C were shifted to 39 C, not only cell growth stopped, but within two to three days the cultures shed a large number of cells into the medium. These cells were nonviable, and shedding stopped only when the number of cells attached had been reduced to that characteristic of the saturation density at 39 C. The remaining attached cells were viable and after the shift to 32 C were again able to grow from the monolayer to high cell densities. This behavior has been compared with that of normal 3T3 and wild-type SV3T3 cells under different conditions. We have also isolated new tsSV3T3 lines, using cells which had been infected with non-mutagenized wild-type SV40. This further demonstrates that the temperature sensitivity of these lines is due to a cellular rather than a viral mutation.     

Multi-Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge of Cynomolgus Macaques Immunized with “Hyperattenuated” SIV Constructs

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Δ5-CMV and Δ6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10⁸ 50% tissue culture infective doses (TCID₅₀) of Δ5-CMV or Δ6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Δ5-CMV vaccine group compared to the Δ6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Δ5-CMV-vaccinated animals (3.7 × 10⁵ copies/ml) was ∼1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 × 10⁴ copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.To date, the most promising approach to inducing sterilizing immunity in the macaque model has been through the use of live attenuated virus (LAV) vaccines based on simian immunodeficiency virus (SIV). A major advantage of an attenuated virus strategy for the development of a human immunodeficiency virus (HIV) vaccine is the ability of attenuated viruses to induce broad and persistent immunity (29, 51). In particular, SIV strains engineered with deletions of nef (SIVΔnef) have afforded the most significant protection upon challenge with pathogenic SIV (13, 14, 29, 60, 65, 72). Numerous SIV-derived live attenuated vaccine models have been developed, many of which employ deletions in the viral accessory genes (3, 12, 14, 15, 25, 29, 30, 53, 64, 72). In many cases, vaccinations have been shown to substantially decrease viral burden during the acute phase of infection, maintain low to undetectable levels of virus during the chronic phase of infection, and limit the progression to AIDS. Although promising, a major caveat to the live attenuated virus vaccine approach is the potential for compensatory reversion and the observations that incompletely attenuated viruses may harbor residual pathogenicity (5, 10, 14). Even SIV constructs containing multiple deletions in nef, vpr, and the negative regulatory element (NRE) can cause AIDS-like disease in adult macaques and particularly in neonates (4, 5, 27, 53). This may be analogous to some human long-term nonprogressors infected by nef-deleted HIV variants in whom a slowly increasing viral burden has been accompanied by disease progression (22, 34, 37). Additional mutations can be engineered into vaccine vectors to generate highly attenuated viruses, but this often comes at the expense of their protective efficacy (8, 23, 30).We previously made two series of novel live attenuated SIV vaccine models (25) in which the simplified SIV constructs retain all the structural viral proteins but have inactivating mutations for all viral accessory genes. These constructs retain significant antigenicity, without the pathogenic effects associated with accessory viral factors, thus limiting or eliminating the potential for reversion (25).Whether administered parenterally or mucosally, conventional challenge trials in macaques have often utilized artificially high single-dose inocula in an effort to ensure that most, if not all, of the naive or placebo-immunized animal subjects become infected following a single exposure. The rationale for using a single massive challenge has been reconsidered in light of the possibility that vaccines with protective efficacy under physiologic challenge conditions may not identified. This practice is now being replaced by an approach designed to better approximate the relatively low in vivo acquisition rates following a single sexual exposure to HIV (21, 45, 69) and should provide a more realistic assessment of vaccine efficacy in “real-world” situations. Importantly, recent studies using this approach have demonstrated viremia of magnitude and kinetics comparable to that seen following single high-dose mucosal inocula (47), and this approach has been used successfully in more recent challenge trials (31, 70). Here we are assessing the safety, immunogenicity, and protective efficacy of two hyperattenuated SIV vaccine candidates following a multi-low-dose intrarectal challenge with highly pathogenic SIVmac239 in the cynomolgus macaque model.SIV-specific humoral immune responses were assessed at various time points postvaccination and postchallenge by Western blotting. Cellular immunogenicity was monitored by evaluation of peripheral T-cell responses (via gamma interferon [IFN-γ] enzyme-linked immunospot [ELISPOT] assay) following stimulation with peptide pools spanning the entire SIVmac239 proteome. The protective efficacy of the different vaccine candidates was assessed by classical endpoints, such as quantitative analysis of plasma viral load, quantitative immunophenotyping of lymphocytes, and clinical markers of disease progression. Even using extremely attenuated SIV constructs with only minimal evidence of replication, a modest immune response that can impact long-term disease progression is generated.     

Integrin α4β7 Is Downregulated on the Surfaces of Simian Immunodeficiency Virus SIVmac239-Infected Cells

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infection results in an early and enduring depletion of intestinal CD4⁺ T cells. SIV and HIV bind integrin α4β7, thereby facilitating infection of lymphocytes that home to the gut-associated lymphoid tissue (GALT). Using an ex vivo flow cytometry assay, we found that SIVmac239-infected cells expressed significantly lower levels of integrin α4β7 than did uninfected cells. This finding suggested a potential viral effect on integrin α4β7 expression. Using an in vitro model, we confirmed that integrin α4β7 was downregulated on the surfaces of SIVmac239-infected cells. Further, modulation of integrin α4β7 was dependent on de novo synthesis of viral proteins, but neither cell death, the release of a soluble factor, nor a change in activation state was involved. Downregulation of integrin α4β7 may have an unappreciated role in the CD4 depletion of the mucosal-associated lymphoid compartments, susceptibility to superinfection, and/or immune evasion.Infection of macaques with simian immunodeficiency virus (SIV) and humans with human immunodeficiency virus (HIV), regardless of the route of transmission, results in early establishment of infection in the gut-associated lymphoid tissue (GALT) (3, 23, 25). Consequently, the CD4⁺ T cells of the GALT are depleted, and intestinal integrity is compromised (4, 21, 37). The mechanism of GALT depletion, as well as the mechanism of viral localization to the GALT, remains poorly understood.GALT localization is mediated, at least in part, by integrins, a large family of “sticky” cell surface proteins (24, 35, 36). Integrins facilitate conversation between the environment and a cell, thereby influencing cellular adhesion, trafficking, proliferation, and signaling. Consequently, numerous viruses, despite having a small number of proteins, have developed mechanisms to exploit integrins and hence cellular processes, in order to facilitate viral replication and immune evasion (17, 24, 34, 36). Examples of such viruses include human cytomegalovirus (39), rotavirus (14), and SIV/HIV (40). One well-studied integrin, α4β7, mediates migration of lymphocytes to the GALT (31, 33). In 2008, Arthos et al. demonstrated that HIV-1 glycoprotein, gp120, binds integrin α4β7, facilitating infection of CD4⁺ T cells and increasing viral replication efficiency (1).Recent in vivo studies have revealed that CD4⁺ T cells expressing high amounts of integrin α4β7 (integrin α4β7 high) are preferentially infected during acute SIV infection (15, 38). In addition, integrin α4β7 high CD4⁺ T cells contain greater than one provirus per cell during peak viral infection, suggesting that the cells are unusually susceptible to superinfection. Unexpectedly, superinfection is not observed in integrin α4β7 high CD4⁺ T cells after peak viral infection (15). Integrin α4β7 high-expressing CD4⁺ T cells are also depleted from the circulation parallel to the loss of intestinal CD4⁺ cells, suggesting a fundamental role for integrin α4β7 in SIV pathogenesis (38). The mechanism underlying the depletion of integrin α4β7 high-expressing cells and whether SIV-infected cells are directly or indirectly involved remain unknown. Thus, understanding the single-cell dynamics of integrin α4β7 during SIV infection may improve our understanding of SIV and HIV pathogenesis and clarify the role of integrin α4β7 signaling in mucosal trafficking.To examine the single-cell dynamics of integrin α4β7 expression during SIV infection, we used a novel, ex vivo, flow cytometry assay (M. Reynolds, unpublished data). We observed that infected, Gag p27⁺ cells expressed significantly (P = 0.0085) lower levels of integrin α4β7 than uninfected, CD4⁺ T cells from the same animal, at the same time point. Thus, we hypothesized that SIV decreases integrin α4β7 expression on the surfaces of virus-infected cells. In vitro, integrin α4β7 expression was downregulated on SIVmac239-infected cells as rapidly as 24 h postinfection. Unexpectedly, integrin α4β7 levels were also perturbed on uninfected cells with an increase in number of cells with intermediate integrin α4β7 expression. The modulation of integrin α4β7 was dependent on de novo synthesis of a viral protein(s), but neither cell death, release of a soluble factor, nor a change in activation state were involved. Combined, this finding suggests an as-yet-unidentified viral effect on integrin α4β7 that may influence depletion of the mucosal associated lymphoid compartments, susceptibility to superinfection, and/or immune evasion during SIV infection.     

Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16⁺ NK cells, delineated a unique dual-function cytotoxic-CD107a⁺/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56⁺ NK cells, α4β7⁺ and α4β7⁻ NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.Human peripheral blood contains two primary subsets of natural killer (NK) cells—a major CD16⁺ CD56^dim subset and a minor CD16⁻ CD56^bright subset. We have defined subsets of rhesus macaque (Macaca mulatta) NK cells and found that, similarly, macaque peripheral blood is dominated by a CD16⁺ CD56⁻ subset but contains two minor CD16⁻ CD56⁺ and CD16⁻ CD56⁻ subpopulations (34). As in humans, macaque CD16⁻ CD56⁺ NK cells are the primary producers of gamma interferon (IFN-γ) and express cell surface markers such as CCR7 and CD62L that mediate homing to lymph nodes, whereas CD16⁺ CD56⁻ NK cells do not express CCR7 or CD62L and primarily mediate cytolytic functions (7, 12, 30, 34). In both humans and macaques, the distribution of NK subsets in peripheral blood is distinct from that observed in lymph nodes and mucosal tissues, where NK cells are primarily CD56⁺ (9, 12, 30, 35).NK cells are important for the control of multiple viral infections, and increasing evidence suggests that NK cells play a significant role in controlling human immunodeficiency virus (HIV) infection (3, 5, 13, 14, 19, 21, 22, 24, 33), as well as simian immunodeficiency virus (SIV) infection of rhesus macaques and sooty mangabeys (6, 16, 26). HIV and SIV primarily replicate in the gut mucosa (18), and although we and others have demonstrated the presence of NK cells in the gastrointestinal tracts of both humans and rhesus macaques (8, 9, 25, 30), the nature of these NK cells is still poorly understood. Interestingly, the integrin α4β7, which directs lymphocyte trafficking to the gut (4), has been shown to be expressed on peripheral blood NK cells in humans and rhesus macaques (11, 27). This finding suggests that the gut mucosa is a site of active NK cell trafficking.Despite the importance of gut-associated lymphoid tissue in HIV/SIV pathogenesis, little is known about the effects of infection on NK cell homing to these tissues. In order to address this deficit, a total of 47 Indian rhesus macaques were studied, 27 of which were SIV naïve and 20 infected with either SIVmac239 (5) or SIVmac251 (15) for more than 150 days (mean duration of infection, 293 days). All animals were housed at the New England Primate Research Center and maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (23a).PBMC isolation and polychromatic flow cytometry staining were carried out using protocols described previously for our laboratory (29, 31); the antibodies used are listed in Table ​Table1.1. NK cells were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34), and CD16 and CD56 expression were used to delineate three primary subsets: CD56⁻ CD16⁺ (CD16⁺), the dominant subset; CD56⁺ CD16⁻ (CD56⁺); and CD56⁻ CD16⁻ (double negative [DN]) (Fig. ​(Fig.11 A). The results of polychromatic flow cytometry analyses demonstrated that α4β7 was expressed at the highest levels on CD16⁺ NK cells and that, while expression on this subset was not altered during SIV infection, α4β7 was significantly upregulated on both CD56⁺ and DN NK cells in SIV-infected animals (Fig. 1B and C). Interestingly, CCR7, which is expressed only on the CD56⁺ and DN NK cell subsets in macaques (30, 34), was concomitantly downregulated on these subsets of NK cells during chronic SIV infection (Fig. ​(Fig.1B).1B). The relationship between the two markers delineated a dichotomous expression pattern between naïve and SIV-infected macaques (Fig. ​(Fig.1D).1D). This dramatic shift in CD56⁺ and DN NK cell trafficking repertoires is likely indicative of increased homing of these NK subsets to the gut coupled to decreased homing to lymph nodes. Also, as shown in Fig. ​Fig.1E,1E, the absolute numbers of both CD16⁺ and DN NK cells increased during chronic SIV infection, resulting in increased absolute numbers of gut-homing α4β7⁺ cells in both subsets. Interestingly, while the absolute numbers of all CD56⁺ NK cells tended to decrease during chronic SIV infection, the absolute numbers of the α4β7⁺ CD56⁺ NK cell subset increased slightly (Fig. ​(Fig.1E,1E, middle panel), further suggesting that multiple subsets of α4β7⁺ NK cells increase during chronic SIV infection.Open in a separate windowFIG. 1.Comparison of α4β7 expression on NK cell subsets in naïve and SIV-infected macaques. (A) Macaque NK cell subsets were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34) and then further delineated into CD56⁺, CD16⁺, and DN subsets. (B) Representative flow cytometry plots of α4β7 and CCR7 expression on NK cell subsets in naïve and SIV-infected macaques. (C) Percentages of α4β7⁺ cells above the background level were compared between naïve and SIV-infected macaques for CD56⁺, CD16⁺, and DN NK subsets. (D) Relationships between α4β7 and CCR7 expression on CD56⁺ and DN NK cells in naïve and SIV-infected macaques. (E) Absolute numbers of total circulating NK cells were determined by using a bead-based flow cytometric assay as described previously (29, 30), and α4β7⁺ NK cell subset counts were extrapolated using these data combined with NK cell frequency data determined by polychromatic flow cytometry (panel A). Horizontal bars indicate median values for 20 to 27 animals. Student''s t tests were used to compare naive and SIV-infected animal groups; P values of >0.05 are considered statistically significant.

TABLE 1.

Antibodies used in polychromatic flow cytometry analyses

Transgenic mice expressing inhibin α-subunit promoter (inhα)/Simian Virus 40 T-antigen (Tag) transgene as a model for the therapy of granulosa cell-derived ovarian cancer

Granulosa cell tumors are rare, 3–7.6% of primary ovarian tumors, although with poor prognosis as the tumor-related mortality rate is 37.3%, with 80% of deaths occurring on recurrence. We have created a transgenic (TG) murine model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian Virus 40 T-antigen (Tag), under the regulation of murine inhibin α-subunit 6 kb promoter (inhα/Tag). Gonadotropin dependent ovarian granulosa cell tumors were formed in females by the age of 5–6 months, with a 100% penetrance. We have successfully used the inhα/Tag model to test different treatment strategies for ovarian tumors. With a gene therapy trial in inhα/Tag mice crossbred with inhα/HSV-TK (herpes simplex virus thymidine kinase) mice (double TG), we proved the principle that targeted expression of HSV-TK gene in gonadal somatic cell tumors enabled tumor ablation by anti-herpes treatment. When we aimed at targeted destruction of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) expressing inhα/Tag tumor cells in vivo by a lytic peptide Hecate-CGβ conjugate, we could successfully kill the tumor cells, sparing the normal cells. We recently found high zona pellucida glycoprotein 3 (ZP3) expression in inhα/Tag granulosa cell tumors, as well as in human granulosa cell tumors. We tested the concept of treating the ovarian tumors of inhα/Tag mice by vaccination against the ectopically expressed ZP3. Immunotherapy with recombinant human (rh) ZP3 was highly successful with no objective side effects in inhα/Tag females, suggesting rhZP3 immunization as a novel strategy for the immunotherapy of ovarian granulosa cell tumors.     

Evidence of Early B-Cell Dysregulation in Simian Immunodeficiency Virus Infection: Rapid Depletion of Na?ve and Memory B-Cell Subsets with Delayed Reconstitution of the Na?ve B-Cell Population

Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus (HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mechanistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodeficiency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very early after infection at the precise time when an effective adaptive immune response is needed.Effective B-cell responses result in the generation of memory B-cell populations which are able to proliferate and produce antibodies that can control primary and secondary insults by microbial pathogens (2). Impaired maturation and timing of B-cell-mediated immune responses result in the production of ineffective antibodies, which are unable to control infection and may result in the persistence of the pathogen (36). Although human immunodeficiency virus (HIV) infection generally elicits high-titer antibodies, virus-specific titers do not correlate with delayed clinical progression, suggesting that antibodies produced during HIV infection are not sufficient to provide long-term viral control (6). Ineffective antibody production in the context of HIV infection could be a result of numerous T-cell and B-cell abnormalities induced either directly or indirectly through infection. B-cell perturbations, characterized during chronic infection, include hypergammaglobulinemia (11, 31), a diminished in vitro response to mitogenic stimulation (10, 37), diminished antibody responses to vaccination (15, 23), and loss of memory B-cell subsets (3, 10, 37). It is highly likely that these B-cell abnormalities are linked with the inability of HIV-infected individuals to form effective antibody responses to HIV and opportunistic pathogens.B-cell perturbations during acute HIV infection may lead to dysfunctions observed during chronic infection. Despite numerous reports that hypothesized that B-cell phenotypic and functional abnormalities arise due to the effects of chronic infection, a limited number of acute infection studies have provided evidence that B-cell dysfunctions may be initiated much earlier. Studies by De Milito et al. and others have reported a decrease in CD27⁺ B cells associated with chronic HIV infection (3, 4, 10-12, 15, 30, 31, 36-38, 40). The reduction of this population may explain the diminished antibody responses to non-HIV antigens present in HIV-infected individuals. However, the mechanism for this loss of memory B cells during chronic infection is unclear. One possibility is that B-cell losses are related to reduced T-cell numbers. In a study by Titanji et al., a strong correlation between the number of CD4 T cells and the percentage of memory B cells was reported in chronic HIV infection (37). Conversely, others have reported that no correlation was found between CD4 numbers and memory B-cell numbers (3, 10). Interestingly, reductions in percentages of B cells, increased expression of Fas on B cells, increased total plasma IgG levels, a decreased percentage of IgM memory B cells, and decreased B-cell responses to antigenic stimulation have been shown to occur within 6 months of HIV infection (36, 37). Disruption of germinal centers in the gut during acute HIV infection may also compromise the humoral immune response (20). While these studies provide insight into virus-induced changes in the B-cell compartment during infection, it is difficult to ascertain precisely when these changes occur, due to limitations in sample size and numbers during this early period of infection. The conflicting reports reflect the high amount of variability present in human HIV infection and illuminate the need for a model to study B-cell populations in which experimental parameters can be more rigorously controlled. An understanding of the effects of HIV on the B-cell population during this critical early phase of infection is needed to determine how the initial interactions between virus and host immune system set the stage for long-term disease progression in the infected host. The simian immunodeficiency virus (SIV)/macaque model provides a system in which to ask these questions.Studies in SIV-infected macaques have demonstrated that the number of total B (CD20⁺) cells in the periphery decreases dramatically during the acute phase of infection (13, 24). The loss of these cells coincides with a similar depletion of peripheral CD4 T cells and is associated with primary viremia. Interestingly, the loss of total B cells is greater in magnitude than the loss of CD4⁺ T cells (24). In order to understand how these cells are being depleted, it is necessary to characterize B-cell subsets during SIV infection in the macaque. The present study was designed to assess phenotypic changes in B-cell numbers during the acute phase of SIV infection, both in the total B-cell population as well as in B-cell subsets. Our results identified early, rapid changes in B-cell subsets that were not apparent in analysis of the total B-cell population. Specifically, we identified a significant depletion from the periphery of both the naïve (CD20⁺ CD27⁻) and memory (CD20⁺ CD27⁺) B-cell populations during acute infection and increased total B-cell population activation that may be related to ineffective antibody production commonly associated with SIV infection. Furthermore, the data demonstrate that measurement of envelope-specific antibody responses was not a sensitive reflection of SIV effects on B-cell subsets. These data provide novel information about the timing and dynamics of phenotypic changes in the B-cell compartment during SIV infection that may be associated with functional changes observed later in chronic infection. These results can be used to tailor therapeutic treatments designed to preserve the B-cell compartment early in SIV/HIV infection.     

14. Studies on Poliomyelitis Virus—Concentration and Purification of the Virus

ABSTRACT

For the purpose of determining the immunogenic potency of polio virus, relatively large amounts of concentrated virus material were prepared which had titres of the order of 10¹⁰ T.C.I.D.jo per ml. These were obtained by pervaporating large quantities of tissue culture fluid containing approximately 10⁶⁵ T.C.I.D.JQ per ml.     

Effect of β-Propiolactone on Sendai Virus

The biological properties (infectivity, hemagglutination, hemolysis, cell fusion, neuraminidase) of Sendai virus were dissociated on the basis of sensitivity to beta-propiolactone, by freeze-thawing, by heating at different temperatures, and by adsorption-elution with formalinized chicken erythrocytes. Possible mechanisms whereby beta-propiolactone selectively destroys viral infectivity are discussed.     

Suppression of Transforming Growth Factor β Receptor 2 and Smad5 Is Associated with High Levels of MicroRNA miR-155 in the Oral Mucosa during Chronic Simian Immunodeficiency Virus Infection

Chronic human immunodeficiency virus and simian immunodeficiency virus (HIV and SIV) infections are characterized by mucosal inflammation in the presence of anti-inflammatory cytokines such as transforming growth factor β (TGFβ). The mechanisms for refractiveness to TGFβ are not clear. Here we show that the expression of microRNA miR-155 was significantly upregulated in the oropharyngeal mucosa during chronic SIV infection and was coincident with downregulation of TGFβ receptor 2 (TGFβ-R2) and SMAD5, key TGFβ signaling genes that harbor putative target sites for miR-155. Ectopic expression of miR-155 in vitro was found to significantly downregulate TGFβ-R2 and Smad5 expression, suggesting a role for miR-155 in the suppression of TGFβ-R2 and SMAD5 genes in vivo. The downregulation of TGFβ signaling genes by miR-155 likely contributes to the nonresponsiveness to TGFβ during SIV infection and may inadvertently aid in increased immune activation during HIV and SIV infections.     

Macaques Vaccinated with Simian Immunodeficiency Virus SIVmac239Δnef Delay Acquisition and Control Replication after Repeated Low-Dose Heterologous SIV Challenge

An effective human immunodeficiency virus (HIV) vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best simian immunodeficiency virus (SIV) vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Δnef and challenged them intrarectally (i.r.) with repeated low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to that for naïve controls (log rank test; P = 0.023). After 10 mucosal challenges, we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these 10 challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than did the naïve controls (Mann-Whitney U test; P = 0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 postinfection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequence analysis of the circulating viruses in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.The goals of any human immunodeficiency virus (HIV) vaccine are both to prevent infection and, if infection occurs, to control virus replication. If vaccinated individuals who become infected are able to reduce virus replication to extremely low or undetectable levels, they will live longer, healthier lives and will be less likely to transmit the virus to others (7, 16, 41). An HIV vaccine that successfully meets these two goals will therefore have a significant impact on slowing the spread of HIV (3).Live-attenuated simian immunodeficiency virus (SIV) vaccines have proven to be universally effective at protecting macaques against homologous virus challenges, regardless of the route of transmission (10, 21, 33, 36, 50, 51). For this reason, live-attenuated SIV vaccines are considered the “gold standard” of protection in the SIV/rhesus macaque model of HIV infection (25). Previously, we and others showed that SIVmac239Δnef-vaccinated animals can reduce plasma virus replication after intravenous (i.v.) inoculation with the uncloned heterologous swarm virus SIVsmE660 (43, 50). This vaccine-induced effect was most pronounced, particularly during acute infection, in animals expressing major histocompatibility complex (MHC) class I alleles (Mamu-A*01, -B*08, and -B*17) previously associated with control of pathogenic SIVmac239 replication (29, 38, 43, 52, 54). Despite these encouraging results for this subset of animals, and in contrast to previous studies using homologous virus challenges, most of the vaccinated animals failed to maintain control of virus replication of the challenge strain during the chronic phase of infection.There are several potential explanations for why SIVmac239Δnef vaccination was not as effective against i.v. exposure to the heterologous challenge virus (1, 43, 50). First, sequence variation between the vaccine and infecting strains may have rendered the vaccine-induced immune responses ineffective at controlling chronic-phase virus replication. Second, unlike the case in homologous SIVmac239 challenge studies using cloned viral stocks, the heterologous SIVsmE660 isolate contains many quasispecies within the inoculum (23, 49). Third, the heterologous challenges were administered i.v., thereby bypassing any potentially protective vaccine-induced immune responses at mucosal surfaces. All of the SIVsmE660 quasispecies in the inoculum therefore had the potential to infect cells and to establish a reservoir of viral diversity. This broad spectrum of viral diversity may have contributed to the decreased efficacy of SIVmac239Δnef-induced immune responses in protecting against heterologous virus replication after a high-dose i.v. challenge.Since a large i.v. dose of multiple quasispecies of heterologous virus might overwhelm any potentially protective vaccine-induced immune responses, we tested the possibility that SIVmac239Δnef vaccination may be more efficacious against a more physiologically relevant low-dose challenge. In the SIV/rhesus macaque model of HIV infection, repeated low doses of pathogenic SIV more accurately reflect human sexual transmission than a single high-dose i.v. challenge does (32). Keele et al. recently established that one to three virus strains typically cross mucosal barriers to establish HIV infections (22). We and others observed similar results using repeated-dose mucosal challenge of macaques (23, 49). This model therefore facilitates the testing of vaccines in a more physiologically relevant manner.