Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism.

The phase III RV144 human immunodeficiency virus (HIV) vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing the HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clade A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: the simultaneous depletion of B and CD8+ cells followed by the intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High-level viremia and CD4+ T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4+ T-cell loss; the partially adapted SHIV had become dual tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5 tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and was pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans.IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in nonhuman primate (NHP) models requires test viruses that allow the evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to nonneutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive-immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed a coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development.

BACKGROUND: We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates. METHODS: Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization. RESULTS: Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals. CONCLUSIONS: The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.

Development of real-time PCR assays for quantitation of simian betaretrovirus serotype-1, -2, -3, and -5 viral DNA in Asian monkeys.

Simian betaretroviruses (SRV), formerly known as simian type D retroviruses, are endemic in many populations of Asian monkeys of the genus Macaca. Asian monkeys have been used extensively as animal models for preclinical HIV vaccine development, therapeutics, and other biomedical studies. SRV infection can sometimes lead to immune deficiency disease, which complicates such studies; thus, it is important to screen for SRV infection and remove infected animals from test populations. Real-time PCR assays were developed to specifically quantify SRV-1/3, SRV-2, and SRV-5 proviral DNA. The SRV provirus copy numbers were standardized relative to real-time PCR measurements of the rhesus macaque albumin gene. The primers and TaqMan probe sequences for the rhesus macaque (Indian origin) albumin gene also detect cynomolgus macaque and rhesus macaque (Chinese origin) albumin genes. The SRV primers and probes were designed to amplify gag gene sequences of SRV-1/3 (GeneBank accession number M11841), SRV-2 (GeneBank accession number M16605), and SRV-5 (GeneBank accession number AF252389). The optimized reactions for detection of each SRV serotype and the macaque albumin gene had amplification efficiencies of greater than 90% with a linear range spanning 1 x 10(1) to 2.5 x 10(6) copies per reaction. The R(2) values of all standard curves were greater than 0.995. Of 40 animals housed in quarantine, four animals were positive for SRV-1/3 with 28, 5450, 9780, and 14,500 copies of provirus per 10(6) PBMCs, and one animal was positive for SRV-2 with provirus copy number of 7790 per 10(6) PBMCs. All of 40 animals appeared to be seronegative and had normal CD4(+) and CD8(+) T-cell counts. These quantitative real-time PCR assays enhance the detection and quantitation of SRV infection and will facilitate the elimination of this virus from macaque colonies.

Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation.

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia. Furthermore, after vaccination with SIV expression plasmids the recall responses to the SIV antigens were very high, indicating that DNA is a strong boost in the presence of antiretroviral treatment in SIV-infected animals. There was substantial animal-to-animal variability in DNA expression, revealed by plasma measurements of IL-15 produced by co-injected IL-15 DNA. IL-15 expression levels correlated with peak immune responses. Electroporation led to an expansion of antigen-specific CD4+ and CD8+ T cells of both central and effector memory phenotype. These results indicate that improved gene delivery and expression by electroporation dramatically increases immunogenicity of DNA vaccines. Electroporation is thus an important method to improve the effectiveness of DNA vaccination.