Induction of inhibitory antibodies to the CCR5 chemokine receptor and their complementary role in preventing SIV infection in macaques.

The seven-transmembrane G-protein-linked CCR5 molecule functions as a major coreceptor for HIV or simian immunodeficiency virus (SIV) infection. Antibodies to CCR5 were studied in rhesus macaques immunized with SIV grown in human CD4(+) T cells. These macaques were completely protected against i.v. challenge with live SIV. Sera from the protected macaques showed significantly greater inhibition of SIV replication (p < 0.001) and macrophage inflammatory protein-1beta-generated CCR5-dependent chemotaxis (p < 0.01) than sera from unprotected macaques, in the absence of significant neutralizing antibodies to SIV. These two functional assays demonstrate serum antibodies to the CCR5 receptors which were specifically inhibited by CCR5-transfected HEK-293 cells. We postulate that anti-CCR5 antibodies may be complementary to beta-chemokines in blocking CCR5 coreceptors to HIV or SIV binding and fusion of CD4(+) cells.     

A novel CD4-conjugated ultraviolet light-activated photocatalyst inactivates HIV-1 and SIV efficiently

In this study, we found that the electric potential derived from the redox reaction of ultraviolet (UV)-illuminated CD4-conjugated titanium dioxide (TiO2) inactivated a wide range of high-titered primary HIV-1 isolates, regardless of virus co-receptor usage or genetic clade. In vitro incubation of HIV-1 isolates with CD4-conjugated TiO2 (CD4-TiO2) followed by UV illumination led to inhibition of viral infectivity in both H9 cells and peripheral blood mononuclear cells as well as to the complete inactivation of plasma virions from HIV-1-infected individuals. Treatment with a newly established extra-corporeal circulation system with the photocatalyst in rhesus macaques completely inactivated plasma virus in the system and effectively reduced the infectious plasma viral load. Furthermore, plasma viremia and infectious viral loads were controlled following a second therapeutic photocatalyst treatment during primary SIV(mac239) infection of macaques. Our findings suggest that this therapeutic immunophysical strategy may help control human immunodeficiency viral infection in vivo.     

Derivation and Biological Characterization of a Molecular Clone of SHIVKU-2 That Causes AIDS, Neurological Disease, and Renal Disease in Rhesus Macaques

Previously, we described the derivation of a pathogenic strain of simian-human immunodeficiency virus (SHIV(KU-2)) consisting of the tat, rev, vpu, and env genes of HIV-1 (strain HXB2) in a genetic background of SIV(mac)239 that causes AIDS and productive infection of the CNS in rhesus macaques (Macca mulatta) (Raghavan et al., 1997, Brain Pathol. 7, 851-861). We report here on the characterization of a molecular clone of SHIV(KU-2), designated SHIV(KU-2MC4), that caused CD4(+) T cell loss as well as neurological and renal disease in macaques. DNA sequence analysis of selected SIV regions of SHIV(KU-2MC4) revealed 10 nucleotide changes in the LTR, whereas Gag, Vif, Vpr, Vpx, and Nef had 1, 1, 1, 2, and 13 predicted amino acid substitutions, respectively, compared to SIV(mac)239. DNA sequence analysis of HIV-1 derived regions of SHIV(KU-2MC4) revealed 2, 1, 2, and 18 predicted amino acid substitutions in the Tat, Rev, Vpu, and Env proteins, respectively, when compared to SHIV-4. Unlike the parental SHIV-4, which is not tropic for macrophages, SHIV(KU-2MC4) replicated efficiently in macrophage cultures as determined by p27 assays. However, despite the numerous changes in the Env protein and newly acquired tropism for macrophages, SHIV(KU-2MC4), like the parental SHIV-4, used CXCR4 exclusively as its coreceptor for entry into susceptible cells. Inoculation of SHIV(KU-2MC4) into two rhesus macaques resulted in severe infection in which the numbers of circulating CD4(+) T cells in the blood declined rapidly by 2 weeks postinoculation and virus producing cells in the peripheral blood mononuclear cells were identified throughout the course of infection. At the time of euthanasia (20 and 22 weeks), both macaques had lost a significant amount of weight and had no circulating CD4(+) T cells. In addition, one macaque developed intension tremors and uncoordinated movements. Virological examination of tissues at necropsy revealed active virus replication in both lymphoid and nonlymphoid tissues such as the lung and brain. Histological examination revealed that the induced immunodeficiency was associated with lymphoid depletion of the lymph nodes and spleen, opportunistic infections, lentiviral encephalitis, and severe glomerulosclerosis of the kidney. This molecular clone will serve as the basis for analyzing the molecular determinants through which SHIV(KU-2) causes severe CD4(+) T cell loss, neurological disease, and SHIV nephropathy in rhesus macaques.     

Expansion of quasispecies diversity but no evidence for adaptive evolution of SHIV during rapid serial transfers among seronegative macaques

Four successive, rapid serial passages of the nonpathogenic, CCR5-tropic simian-human immunodeficiency virus SHIV(SF162) in rhesus macaques resulted in an increase in acute plasma viremia with each passage and the emergence of a pathogenic isolate SHIV(SF162P3) in one of the passage three transfer animals (macaque T353). To explore the mechanism(s) underlying increased virulence of SHIV(SF162) upon in vivo passage, the evolution of the HIV-1 envelope gene was characterized in plasma and PBMC samples obtained from animals before (week 1) and after (week 3) the time of virus transfer. We found no evidence in support of adaptive evolution of the HIV gp120 during rapid serial passage; however, the animals which later received passage virus had more diverse quasispecies. SHIV(SF162P3)-like gp120 sequences were first detected in macaque T353 at week 6, after seroconversion. These sequence changes increased in frequency and number at later time points. The first sequence change conferred neutralization escape but not an increase in viral infectivity that could account for the apparent increase in replicative capacity of the later passage viruses. Collectively, our data argue against any host-specific adaptation of the HIV-1 envelope gp120 as the basis for the generation of more aggressive SHIV variants during rapid serial transfers in seronegative macaques, and support the model of quasispecies diversity as a predictor of pathogenesis. Envelope sequence changes accumulate principally in response to immune pressure exerted by the host, generating viral variants that can persist in the presence of a strong host immune response.     

HIV-1 prophylactic vaccine comprised of topical DermaVir prime and protein boost elicits cellular immune responses and controls pathogenic R5 SHIV162P3

Topical DNA vaccination (DermaVir) facilitates antigen presentation to naive T cells. DermaVir immunization in mice, using HIV-1 Env and Gag, elicited cellular immune responses. Boosting with HIV-1 gp120 Env and p41 Gag augmented Th1 cytokine levels. Intramuscular DNA administration was less efficient in priming antigen-specific cytokine production and memory T cells. In rhesus macaques, DermaVir immunization induced Gag- and Env-specific Th1 and Th2 cytokines and generation of memory T cells. Boosting of DermaVir-primed serum antibody levels was noted following gp140(SHIV89.6P)/p27(SIV) immunization. Rectal challenge with pathogenic R5-tropic SHIV162P3 resulted in control of plasma viremia (4/5 animals) that was reflected in jejunum, colon and mesenteric lymph nodes. An inverse correlation was found between Gag- and Env-specific central memory T cell responses on the day of challenge and plasma viremia at set point. Overall, the topical DermaVir/protein vaccination yields central memory T cell responses and facilitates control of pathogenic SHIV infection.     

CD4+ lymphocytopenia in acute infection of Asian macaques by a vaginally transmissible subtype-C,CCR5-tropic Simian/Human Immunodeficiency Virus (SHIV)

An R5-tropic SHIV(CHN19P4) was previously generated using a primary HIV-1 subtype-C envelope. We have further characterized this SHIV in two species of macaques. To determine whether this isolate is transmissible vaginally, female pig-tailed macaques were inoculated with 2 x 10(3) TCID50 of SHIV(CHN19P4) by the vaginal route. Animals became infected with a high peak plasma viremia (>10(7) viral copies/mL) and rapid seroconversion. The viremia was accompanied by CD4+ lymphocytopenia in the gut lamina propria lymphocyte (LPL) population. Comparable CD4+ T-cell loss was not seen in peripheral blood and colonic lymph nodes. These findings demonstrate a unique R5-tropic SHIV that can be used to study envelope-related issues in vaginal transmission of the most prevalent subtype of HIV-1. We also found that rhesus macaques intravenously inoculated with 1 x 10(3) TCID50 of SHIV(CHN19P4) became infected and showed CD4+ lymphocytopenia in the gut LPL population. Despite inactivation of the vpu gene in SHIV(CHN19P4), the virus appears to target mainly gut-associated lymphoid tissues during the initial stage of infection as has been described for SHIV(SF162P), another R5-tropic (subtype B) recombinant virus. Our data indicate that the R5-mediated CD4+ lymphocytopenia in the gut is likely independent of HIV-1 genotypes and of the function of vpu at the acute phase of viral infection.     

Pathogenic determinants of the mucosally transmissible CXCR4-specific SHIV(SF33A2) map to env region.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established method to study AIDS pathogenesis and is increasingly used to assess the efficacy of vaccine and antiviral candidates. For these reasons, a detailed understanding of those molecular determinants, which confer pathogenic potential to SHIV viruses, should assist in both rational experimental design and interpretation of results. In this report, we describe the development and in vivo characterization of a pathogenic molecular clone, SHIVSF33A2, which contains an envelope sequence derived from the CXCR4-dependent isolate, HIV-1SF33. Proviral DNA, amplified from a rhesus macaque infected with the pathogenic isolate SHIVSF33A, was substituted into the corresponding region of the parental, nonpathogenic SHIVSF33 genome creating the molecular clone SHIVSF33A2. Coreceptor specificity of SHIVSF33A2 was determined to be CXCR4 specific. Naive rhesus macaques were productively infected after a single exposure to cell-free SHIVSF33A2 by either the intravenous (IV) or intravaginal (IVAG) routes. Animals infected with SHIVSF33A2 suffered a severe loss of peripheral CD4+ T cells and high acute plasma viremia with development of simian AIDS 9 months after inoculation. Sequence analysis identified 25 discreet amino acid changes within the V1-V5 regions of the envelope protein when compared with the nonpathogenic parental virus. These data indicate that domains within the HIV-1 envelope protein are sufficient to define pathogenic potential in the context of the SIVmac239 genome.     

Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV(KU2) infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-gamma-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activity against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV(KU2). Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-gamma production, higher levels of vaccine-specific IFN-gamma producing CD4(+) cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.     

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework

Summary.  To establish simian/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among subtype B envelope, four subtype E V3 sequences were substituted into SHIV_MD14, a SHIV clone bearing an envelope derived from a CXCR4 (X4)/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an only V3-chimera clone capable of replicating in human and macaque peripheral blood mononuclear cells (PBMCs), was propagated in pig-tailed macaque PBMCs and in cynomolgus macaque splenic mononuclear cells. The propagated virus stocks were intravenously inoculated into respective macaque species. SHIV-TH09V3 infected both macaque species as shown by plasma RNA viremia, isolated viruses from PBMCs and plasma, and antibody production against viral proteins. To assess how the substituted V3 sequence affected coreceptor usage, SHIV-TH09V3 stocks propagated in vitro and after isolation from macaques were verified for their corecepor usage by GHOST cells assay. SHIV-TH09V3 maintained R5-tropic phenotype both in vitro and after isolation from macaques, in contrast to the X4/R5-dual tropic SHIV_MD14. This indicates the substituted V3 sequence among the backbone of SHIV_MD14 governs coreceptor usage. Future study of infecting macaques with SHIV-TH09V3 and SHIV_MD14 will focus on differences of the outcome caused by the different V3 sequences in connection with coreceptor usage. Received July 30, 2002; accepted November 13, 2002 Published online March 21, 2003     

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to na?ve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.     

Nonpathogenic CCR2-tropic SIVrcm after serial passage and its effect on SIVmac infection of Indian rhesus macaques

The natural host of SIVrcm is the red-capped mangabey (Cercocebus torquatus torquatus). Although this virus infects macaques and human PBMCs, its pathogenic potential is unknown. We serially passaged SIVrcm through 9 rhesus macaques to assess its potential for virulence. SIVrcm infected all macaques with peak viremia 2 weeks postinfection yet viral loads decreased to undetectable levels about one month after inoculation. Remarkably, SIVrcm replication and virulence did not increase following 7 serial passages. While CD4+ T cells in the gut were decreased in early infection, proportions of memory CD4+CCR5+ T cells were not affected. Three SIVrcm-infected macaques were subsequently challenged with SIVmac251 to assess the potential for superinfection. Interestingly, animals previously infected with SIVrcm had 100 fold lower levels of SIVmac251 in plasma compared to naive animals inoculated with SIVmac251. These results suggest that SIVrcm is nonpathogenic and may be useful for examining effective immune responses in SIV infection.     

Neuropathogenesis of Chimeric Simian/Human Immunodeficiency Virus infection In Pig-tailed and Rhesus Macaques

We recently reported that a chimeric simian/human immunodeficiency virus (SHIV_KU-1) developed in our laboratory caused progressive depletion of CD4⁺T lymphocytes and AIDS within 6 months of inoculation into pig-tailed macaques (M.nemestrina). None of the pig-tailed macaques showed productive SHIV infection in the central nervous system (CNS). In this report, we show that by further passage of the pathogenic virus in rhesus macaques [M. mulatta], we have derived a new strain of SHIV (SHIV_KU-2) that has caused AIDS and productive CNS infection in 3 of 5 rhesus macaques infected with the virus. Productive replication of SHIV in the CNS was clearly shown by high infectivity titers and p27 protein levels in brain homogenates, and in 2 of the 3 rhesus macaques this was associated with disseminated, nodular, demyelinating lesions, including focal multinucleated giant cell reaction, largely confined to the white matter. These findings were reminiscent of HIV-1 associated neurological disease, and our immunohistochemical and in situ hybridization data indicated that the neuropathological lesions were associated with the presence of SHIV-specific viral antigens and nucleic acid respectively. However, the concomitant reactivation of opportunistic infections in these macaques suggested that such pathogens may have influenced the replication of SHIV in the CNS, or modified the neuropathological sequelae of SHIV infection in the rhesus species, but not in pig-tailed macaques. Our findings in the two species of macaques highlight the complexities of lentiviral neuropathogenesis, the precise mechanisms of which are still elusive.     

Murine models for HIV vaccination and challenge

HIV-1 only infects humans and chimpanzees. SIV or SHIV are, therefore, used as models for HIV in rhesus, cynomologus and pigtail macaques. Since conducting experiments in primate models does not fully mimic infection or vaccination against HIV-1 and is expensive, there is a great need for small-animal models in which it is possible to study HIV-1 infection, immunity and vaccine efficacy. This review summarizes the available murine models for studying HIV-1 infection with an emphasis on our experience of the HIV-1-infected-cell challenge as a model for evaluating candidate HIV-1 vaccines. In the cell-based challenge model, several important factors that, hopefully, can be related to vaccine efficacy in humans were discovered: the efficiency of combining plasmid DNA representing several of the viral genes originating from multiple clades of HIV-1, the importance of adjuvants activating innate and induced immunity and the enhanced HIV eradication by drug-conjugated antibody.     

Phenotypic and kinetic analysis of effective simian-human immunodeficiency virus-specific T cell responses in DNA--and fowlpox virus-vaccinated macaques

Although T cell immunity is important in the control of HIV-1 infection, the characteristics of effective HIV-specific T cell responses are unclear. We previously observed protection from virulent SHIV challenges in macaques administered priming with DNA vaccines and boosting with recombinant fowlpox viruses expressing shared SIV Gag antigens. We therefore performed a detailed kinetic and phenotypic study of the T cell immunity induced by these vaccines prior to and following SHIV challenge utilizing intracellular cytokine staining. Pigtail macaques vaccinated intramuscularly with DNA/recombinant fowlpox virus exhibited a coordinated induction of first Gag-specific CD4 T cell responses and then a week later Gag-specific CD8 T cell responses following the fowlpox virus boost. Overall, the magnitude and timing of the peak CD8 T cell responses following challenge was significantly associated with reductions in SHIV viremia following pathogenic challenge. After pathogenic lentiviral challenge, virus-specific effector memory T cells derived from animals controlling SHIV infection recognized a broad array of epitopes, expressed multiple effector cytokines and rapidly recognized virus-exposed cells ex vivo. These results shed light on some of the requirements for T cells in the control of pathogenic lentiviral infections.     

Replicating adenovirus HIV/SIV recombinant priming alone or in combination with a gp140 protein boost results in significant control of viremia following a SHIV89.6P challenge in Mamu-A*01 negative rhesus macaques

Previously, replicating adenovirus type 5 host range (Ad5hr)-HIV/SIV recombinant priming in combination with SIV envelope boosting, resulted in significant, durable protection in 39% of rhesus macaques after SIVmac251 challenge. Both Env-specific antibody mediating ADCC, and cellular immunity correlated with protection. Here we evaluate the relative immunogenicities of novel HIV proteins and their contribution to protection in a SHIV89.6P model. All groups were primed with Ad-HIVenv89.6P, SIVgag239, and SIVnef239 recombinants. One group was not boosted, one received HIV89.6Pgp140DeltaCFI protein, and one a novel HIV-1 poly-peptide "peptomer". The HIV89.6Pgp140DeltaCFI protein in adjuvant strongly boosted Env-specific antibody and memory T cell responses in blood and tissue, resulting in significant reductions in acute and set point viremia. Macaques not boosted, showed a significant reduction in set point viremia, a full 32 weeks after the last Ad priming immunization. The HIV peptomer-boosted group showed a trend toward chronic viremia reduction, but was not protected.     

Functional genomics analyses of differential macaque peripheral blood mononuclear cell infections by human immunodeficiency virus-1 and simian immunodeficiency virus

The pathogenicity of the primate lentiviruses, human, and simian immunodeficiency viruses, is host-specific. Previous studies indicated that the highly pathogenic human lentivirus HIV-1 has markedly reduced pathogenicity compared to the pathogenic simian lentivirus SIV in pigtail macaques (Macaca nemestrina). We therefore hypothesized that the pigtail macaque peripheral blood mononuclear cells (mPBMCs) would respond differently to infections of HIV-1 and pathogenic SIV. To elucidate the cellular responses to the infections of HIV-1 and SIV, we infected mPBMC with these two viruses. Like infections in vivo, HIV-1 and SIV demonstrated distinct replication kinetics in mPBMCs, with HIV-1 replicating at significantly lower levels. Similarly, gene expression profiling facilitated by macaque-specific oligonucleotide microarrays also revealed distinct expression patterns of genes between the HIV-1- and SIV-infected mPBMCs; in particular, genes associated with the antigen presentation, T cell receptor, ERK/MAPK signaling, Wnt/beta-catenin signaling, and natural killer cell signaling pathways were differentially regulated between these two viruses. Most interestingly, despite the lower levels of replication, HIV-1 triggered a more robust regulation of immune response genes early after infection; the converse was true in SIV-infected mPBMCs. Our results therefore suggest that macaques may be controlling the infection of HIV-1 at an early stage through coordinated regulation of host defense pathways.