Mother-to-infant transmission of SIV via breast-feeding in rhesus macaques.

To decipher the mechanisms involved in oral transmission of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) through breast-feeding, we have developed an animal model using SIV-infected lactating rhesus macaques (Macaca mulatta) and their infants. Five of eight macaque infants became infected during a 10-month study course after SIV inoculation of lactating dams. In a second study, three of four chronically infected female macaques transmitted virus to their infants through breast-feeding within 4 months of birth. Transmission of virus to infants did not correlate with viral loads in either milk or plasma. Infants were infected with homogeneous virus populations, while milk samples near the time of transmission were more diverse. These studies suggest that specific viral phenotypes are selectively transmitted through breast-feeding.

Production and characterization of SIV envelope-specific rhesus monoclonal antibodies from a macaque asymptomatically infected with a live SIV vaccine.

Five rhesus monoclonal antibodies (RhMAbs) were produced by rhesus EBV transformation of peripheral blood B cells from a rhesus macaque that had been asymptomatically infected with an attenuated, macrophage-tropic SIV strain, 17E-Cl. These MAbs recognized conformation-dependent epitopes on SIV gp120 and could not be mapped using synthetic peptides. All five RhMAbs were able to neutralize the vaccine strain and a heterologous isolate, SIV/DeltaB670. The RhMAbs did not cross-react with HIV-2; by contrast, four human MAbs derived from an HIV-2-infected person were broadly cross-reactive with both SIV and HIV-2 gp120s. Cross-competition analysis indicated that the five RhMAbs could be placed in two groups recognizing two nonoverlapping epitopes; while the HMAbs were placed in two additional competition groups. Binding of the three group I RhMAbs (1.7F, 3.11B, and 1.10A) as well as HMAb 17A was shown to be sensitive to specific amino acid alterations in V4 occurring in natural env variants. The results of this study demonstrate that RhEBV transformation provides a means to probe rhesus antibody responses to SIV infection at the monoclonal level. RhMAbs will allow structural and functional studies of envelope glycoprotein determinants that elicit protective immune responses against SIV.

Tracking members of the simian immunodeficiency virus deltaB670 quasispecies population in vivo at single-cell resolution.

Genetically distinct lentiviruses constitute a quasispecies population that can evolve in response to selective forces. To move beyond characterization of the population as a whole to the behavior of individual members, we devised an in situ hybridization approach that uses genotype-specific probes. We used probes that detect simian immunodeficiency viruses (SIV) that differ in sequence in the V1 region of the surface envelope glycoprotein (env) gene to investigate the replication and cellular tropisms of four viral variants in the tissues of infected rhesus macaques. We found that the V1 genotypic variants replicated in spatially defined patterns and to different extents at each anatomic site. The two variants that replicated most extensively in animals with AIDS were detected in both macrophages and T lymphocytes in tissues. By extension of this approach, it will be possible to investigate the role of individual lentiviruses in a quasispecies in pathogenesis and to evaluate the effects of antiviral or immunotherapeutic treatment on select members of a quasispecies.

Pathogenesis of SIV encephalitis. Selection and replication of neurovirulent SIV.

To investigate the viral and host factors that contribute to neurological disease, nine macaques were intravenously co-inoculated with SIV/DeltaB670, a primary isolate of SIV consisting of at least 21 different genotypes, and SIV/17E-Fr, a neurovirulent recombinant clone. CD4+ cell counts and antigenemia were measured throughout infection. The SIV env V1 region was amplified from brain and peripheral blood mononuclear cell DNA to compare the genotypes present in brain and blood. Seven of the 9 macaques (78%) developed typical SIV-associated neurological lesions classified as severe (4 macaques), moderate (2 macaques), or mild (1 macaque) with a mean time to euthanasia of 7 months. Macaques with severe neurological lesions progressed more rapidly, with a mean time to euthanasia of 3-6 months. SIV/17E-Fr was detected in brain homogenates from all four macaques with severe encephalitis, and in three of the four, SIV/17E-Fr was the only genotype identified in the central nervous system. Macaques with less severe or no neurological lesions usually had one of various genotypes of SIV/DeltaB670 in brain. A variety of genotypes of SIV/DeltaB670 and SIV/17E-Fr were detected in peripheral blood mononuclear cells throughout infection. Macaques with severe neurological lesions had the most precipitous declines in CD4+ cell counts, the highest levels of antigenemia, and the greatest expression of viral RNA and protein in the central nervous system. Macaca nemestrina were more likely to develop severe neurological lesions than M. mulatta or M. fascicularis (P = 0.048). This study demonstrated that neurovirulent strains within the virus swarm can selectively enter and become established in the central nervous system and that the neurological lesions that develop are correlated with the development of host immunosuppression. The species differences in severity of neurological lesions seen in this study suggest that host factors are also important in determining the outcome of lentiviral infection.

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.

Genotypic selection of simian immunodeficiency virus in macaque infants infected transplacentally.

To understand viral and host factors that contribute to transplacental transmission of human immunodeficiency virus, we developed an animal model using pregnant female macaques infected with simian immunodeficiency virus (SIV). Pregnant females were inoculated intravenously during midgestation with either a well-characterized primary isolate of SIV (SIV/DeltaB670) or a combination of SIV/DeltaB670 and the macrophage-tropic molecular clone SIV/17E-Fr. The viral genetic diversity in five infected female macaques and their in utero-infected infants was analyzed. All of the mothers harbored a genetically diverse virus population at parturition, whereas a single genotype from the maternal quasispecies was identified in the infants at birth. Only one of two variants was found in the infants: SIV/17E-Fr (two cases) or a genotype contained within the SIV/DeltaB670 quasispecies (three cases). The macrophage-tropic properties of both transmitted genotypes were suggested by productive replication in primary rhesus macrophage cultures in vitro and the clonal presence in central nervous system tissue of infected monkeys with encephalitis. These observations provide compelling evidence for both genotypic and phenotypic selection in transplacental transmission of SIV and suggest a critical role for macrophages in fetal infection in utero.

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.

A general model for the surface glycoproteins of HIV and other retroviruses.

A hypothetical model of the surface (SU) glycoproteins of human immunodeficiency virus (HIV) and other retroviruses is proposed. The model is based on repetition of a limited number of sequence motifs conserved within the virus family; similarities in biological, immunological, or genetic properties; as well as the tendency for regions of dissimilar sequence to share protein structures predicted by computer algorithms. It is proposed that the protein consists of three structural and functional domains interspersed by relatively conserved interdomain regions. For each retrovirus, these amino-terminal, central, and carboxy-terminal domains may play different roles in binding, postbinding events, and the immune response to viral infection.