Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.     

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.     

Identification of viral determinants of macrophage tropism for simian immunodeficiency virus SIVmac.

Simian immunodeficiency virus (SIV), a lymphocytopathic lentivirus, induces an AIDS-like disease in rhesus macaques (Macaca mulatta). A pathogenic molecular clone of rhesus macaque SIV (SIVmac), SIVmac-239, replicates and induces cytopathology in T lymphocytes but is restricted for replication in macrophages. In contrast, a nonpathogenic molecular clone of SIVmac, SIVmac-1A11, replicates and induces syncytia (multinucleated giant cells) in cultures of both T lymphocytes and macrophages. SIVmac-1A11 does not cause disease in macaques. To map the viral determinants of macrophage tropism, reciprocal recombinant genomes were constructed between molecular clones of SIVmac-239 and SIVmac-1A11. Infectious recombinant viruses were rescued by transfection of cloned viral genomes into permissive lymphoid cells. Analysis of one pair of reciprocal recombinants revealed that an internal 6.2-kb DNA fragment of SIVmac-1A11 was necessary and sufficient for both syncytium formation and efficient replication in macrophages. This region includes the coding sequences for a portion of the gag gene, all of the pol, vif, vpr, and vpx genes, the first coding exons of tat and rev, and the external env glycoprotein gp130. Thus, the transmembrane glycoprotein of env, the nef gene, the second coding exons of tat and rev, and the long terminal repeats are not essential for in vitro macrophage tropism. Analysis of additional recombinants revealed that syncytium formation, but not virus production, was controlled by a 1.4-kb viral DNA fragment in SIVmac-1A11 encoding only the external env glycoprotein gp130. Thus, gp130 env of SIVmac-1A11 is necessary for entry of virus into macrophages but is not sufficient for a complete viral replication cycle in this cell type. We therefore conclude that gp130 env and one or more genetic elements (exclusive of the long terminal repeats, transmembrane glycoprotein of env, and second coding exons of tat and rev, and nef) are essential for a complete replication cycle of SIVmac in rhesus macaque macrophages.     

Immunization with a live, attenuated simian immunodeficiency virus vaccine leads to restriction of viral diversity in Rhesus macaques not protected from pathogenic challenge

Rhesus macaques immunized with simian immunodeficiency virus SIVmac239Deltanef but not protected from SIVmac251 challenge were studied to determine the genetic and biological characteristics of the breakthrough viruses. Assessment of SIV genetic diversity (env V1-V2) revealed a reduction in the number of viral species in the immunized, unprotected macaques, compared to the number in nonimmunized controls. However, no evidence for selection of a specific V1-V2 genotype was observed, and biologically cloned isolates from the animals with breakthrough virus were similar with respect to replication kinetics and coreceptor use in vitro.     

Role of the SH3-Ligand Domain of Simian Immunodeficiency Virus Nef in Interaction with Nef-Associated Kinase and Simian AIDS in Rhesus Macaques

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P¹⁰⁴A/P¹⁰⁷A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.     

Experimental coinfection of rhesus macaques with rhesus cytomegalovirus and simian immunodeficiency virus: pathogenesis

Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.     

nef gene is required for robust productive infection by simian immunodeficiency virus of T-cell-rich paracortex in lymph nodes

The pathogenesis of AIDS virus infection in a nonhuman primate AIDS model was studied by comparing plasma viral loads, CD4(+) T-cell subpopulations in peripheral blood mononuclear cells, and simian immunodeficiency virus (SIV) infection in lymph nodes for rhesus macaques infected with a pathogenic molecularly cloned SIVmac239 strain and those infected with its nef deletion mutant (Deltanef). In agreement with many reports, whereas SIVmac239 infection induced AIDS and depletion of memory CD4(+) T cells in 2 to 3 years postinfection (p.i.), Deltanef infection did not induce any manifestation associated with AIDS up to 6.5 years p.i. To explore the difference in SIV infection in lymphoid tissues, we biopsied lymph nodes at 2, 8, 72, and 82 weeks p.i. and analyzed them by pathological techniques. Maximal numbers of SIV-infected cells (SIV Gag(+), Env(+), and RNA(+)) were detected at 2 weeks p.i. in both the SIVmac239-infected animals and the Deltanef-infected animals. In the SIVmac239-infected animals, most of the infected cells were localized in the T-cell-rich paracortex, whereas in the Deltanef-infected animals, most were localized in B-cell-rich follicles and in the border region between the paracortex and the follicles. Analyses by double staining of CD68(+) macrophages and SIV Gag(+) cells and by double staining of CD3(+) T cells and SIV Env(+) cells revealed that SIV-infected cells were identified as CD4(+) T cells in either the SIVmac239 or the Deltanef infection. Whereas the many functions of Nef protein were reported from in vitro studies, our finding of SIVmac239 replication in the T-cell-rich paracortex in the lymph nodes supports the reported roles of Nef protein in T-cell activation and enhancement of viral infectivity. Furthermore, the abundance of SIVmac239 infection and the paucity of Deltanef infection in the T-cell-rich paracortex accounted for the differences in viral replication and pathogenicity between SIVmac239 and the Deltanef mutant. Thus, our in vivo study indicated that the nef gene enhances SIV replication by robust productive infection in memory CD4(+) T cells in the T-cell-rich region in lymphoid tissues.     

In Vivo Replication Capacity Rather Than In Vitro Macrophage Tropism Predicts Efficiency of Vaginal Transmission of Simian Immunodeficiency Virus or Simian/Human Immunodeficiency Virus in Rhesus Macaques

We used the rhesus macaque model of heterosexual human immunodeficiency virus (HIV) transmission to test the hypothesis that in vitro measures of macrophage tropism predict the ability of a primate lentivirus to initiate a systemic infection after intravaginal inoculation. A single atraumatic intravaginal inoculation with a T-cell-tropic molecular clone of simian immunodeficiency virus (SIV), SIVmac239, or a dualtropic recombinant molecular clone of SIV, SIVmac239/1A11/239, or uncloned dualtropic SIVmac251 or uncloned dualtropic simian/human immunodeficiency virus (SHIV) 89.6-PD produced systemic infection in all rhesus macaques tested. However, vaginal inoculation with a dualtropic molecular clone of SIV, SIVmac1A11, resulted in transient viremia in one of two rhesus macaques. It has previously been shown that 12 intravaginal inoculations with SIVmac1A11 resulted in infection of one of five rhesus macaques (M. L. Marthas, C. J. Miller, S. Sutjipto, J. Higgins, J. Torten, B. L. Lohman, R. E. Unger, H. Kiyono, J. R. McGhee, P. A. Marx, and N. C. Pedersen, J. Med. Primatol. 21:99–107, 1992). In addition, SHIV HXBc2, which replicates in monkey macrophages, does not infect rhesus macaques following multiple vaginal inoculations, while T-cell-tropic SHIV 89.6 does (Y. Lu, P. B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045–3050, 1996). These results demonstrate that in vitro measures of macrophage tropism do not predict if a SIV or SHIV will produce systemic infection after intravaginal inoculation of rhesus macaques. However, we did find that the level to which these viruses replicate in vivo after intravenous inoculation predicts the outcome of intravaginal inoculation with each virus.     

Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

Retroviral recombination in vivo: viral replication patterns and genetic structure of simian immunodeficiency virus (SIV) populations in rhesus macaques after simultaneous or sequential intravaginal inoculation with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef

To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Deltavpx/Deltavpr and then with SIVmac239Deltanef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Deltanef when plasma SIVmac239Deltavpx/Deltavpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Deltanef, and then with SIVmac239Deltavpx/Deltavpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.     

Variability of bio-clinical parameters in Chinese-origin Rhesus macaques infected with simian immunodeficiency virus: a nonhuman primate AIDS model

Background

Although Chinese-origin Rhesus macaques (Ch RhMs) infected with simian immunodeficiency virus (SIV) have been used for many years to evaluate the efficacy of AIDS vaccines and therapeutics, the bio-clinical variability of such a nonhuman primate AIDS model was so far not established.

Methodology/Principal Findings

By randomizing 150 (78 male and 72 female) Ch RhMs with diverse MHC class I alleles into 3 groups (50 animals per group) challenged with intrarectal (ir) SIVmac239, intravenous (iv) SIVmac239, or iv SIVmac251, we evaluated variability in bio-clinical endpoints for 118 weeks. All SIV-challenged Ch RhMs became seropositive for SIV during 1–2 weeks. Plasma viral load (VL) peaked at weeks 1–2 and then declined to set-point levels as from week 5. The set-point VL was 30 fold higher in SIVmac239 (ir or iv)-infected than in SIVmac251 (iv)-infected animals. This difference in plasma VL increased overtime (>100 fold as from week 68). The rates of progression to AIDS or death were more rapid in SIVmac239 (ir or iv)-infected than in SIVmac251 (iv)-infected animals. No significant difference in bio-clinical endpoints was observed in animals challenged with ir or iv SIVmac239. The variability (standard deviation) in peak/set-point VL was nearly one-half lower in animals infected with SIVmac239 (ir or iv) than in those infected with SIVmac251 (iv), allowing that the same treatment-related difference can be detected with one-half fewer animals using SIVmac239 than using SIVmac251.

Conclusion/Significance

These results provide solid estimates of variability in bio-clinical endpoints needed when designing studies using the Ch RhM SIV model and contribute to the improving quality and standardization of preclinical studies.     

Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.     

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with simian immunodeficiency virus is predictive of disease outcome and vaccine efficacy.

Competitive PCR was used to evaluate the expression of cytokine, granzyme B, and chemokine genes in lymph nodes of macaques recently infected with the simian immunodeficiency virus (SIV) pathogenic molecular clone SIVmac239 (n = 16), the nonpathogenic vaccine strain SIVmac239 delta nef (n = 8), and the nonpathogenic molecular clone SIVmac1A11 (n = 8). For both SIVmac239 and its nef-deleted derivative, strong expression was observed as early as 7 days postinfection for interleukin 1beta (IL-1beta), IL-6, tumor necrosis factor alpha, gamma interferon, and IL-13. The levels of gene induction were equally intense for both viruses despite a lower viral load for SIVmac239 deltanef compared with that for SIVmac239. However, the nature of the cytokine network activation varied with the viral inocula. Primary infection with SIVmac239 was characterized by a higher level of IL-4, IL-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES gene expression and a lower level of IL-12 and granzyme B gene expression compared with infection with SIVmac239 delta nef. Thus, infection with nef-deleted SIV was associated with a preferential Th1 versus Th2 pattern of cytokine production. Infection with SIVmac1A11 was characterized by a delayed immune response for all markers tested. The unique patterns of cytokine and chemokine gene expression in lymph nodes correlated nicely with the pathogenic potential of the SIV strains used as well as with differences in their ability to serve as protective vaccines.     

vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys.

In previous experiments, animals infected with SIVmac239 containing a point mutation in the vpr and nef genes developed AIDS-like symptoms after early reversion of the vpr and nef genes. Here we show that two animals in which the nef gene but not the vpr gene had reverted in the first few months did not develop disease during a 3-year observation period even after reversion to a functional vpr gene 70 weeks postinfection. To study the influence of a stable vpr mutation on virus load and pathogenesis, a 43-bp deletion was introduced into the vpr gene of SIVmac239on, a nef-open mutant of SIVmac239. Four rhesus monkeys were inoculated with the vpr deletion mutant (SIV delta vpr), and two control animals were infected with SIVmac239on. Both control animals had persistent antigenemia, high cell-associated virus loads, and elevated neopterin levels. They had to be euthanized 20 and 30 weeks postinfection because of AIDS-related symptoms. However, all four rhesus monkeys inoculated with SIV delta vpr showed only transiently detectable antigenemia. The cell-associated virus loads were high in three of the four animals. Two animals with AIDS-like symptoms had to be euthanized 71 and 73 weeks postinfection. The two remaining monkeys infected with SIV delta vpr were still alive 105 weeks postinfection. In contrast to the SIVmac239on-infected animals, SIV delta vpr-infected animals had strong humoral immune responses and intermittent cellular immune responses to SIV antigens. Our data show that a functional vpr gene is not necessary for pathogenesis. However, vpr-deficient SIVmac239 variants might be slightly attenuated, allowing some animals to resist progression to disease for an extended period of time.     

In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxphi) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (DeltaGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239DeltaGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239DeltaGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxphi signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.     

Neutralizing Antibodies in Sera from Macaques Immunized with Attenuated Simian Immunodeficiency Virus

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.     

Characterization of SIV-specific CD4+ T-helper proliferative responses in macaques immunized with live-attenuated SIV

Analysis of immune responses generated by live-attenuated simian immunodeficiency virus (SIV) strains may provide clues to the mechanisms of protective immunity induced by this approach. We examined SIV-specific T-helper responses in macaques immunized with the live-attenuated SIV strains SIVmac239deltanef and SIVmac239delta3. Optimization of the concentration and duration of antigenic stimulation resulted in the detection of relatively strong SIV-specific proliferative responses, with peak stimulation indices of up to 84. SIV-specific proliferative responses were mediated by CD4+ T cells and were major histocompatibility (MHC) class II restricted. Limiting dilution analysis revealed SIV-specific T-helper precursor frequencies of up to 96 per 10(6) peripheral blood mononuclear cells (PBMC). Intracellular flow-cytometric analysis demonstrated the production of interleukin (IL)-2, interferon (IFN)-gamma, RANTES and macrophage inhibitory protein-1alpha (MIP-1alpha) by T lymphocytes from SIVmac239deltanef-vaccinated animals following SIV p55 stimulation. Induction of strong SIV-specific T-helper responses by live-attenuated SIV vaccines may play a role in their ability to induce protective immunity.