Viral dynamics of early HIV infection in neonatal macaques after oral exposure to HIV-2287: an animal model with implications for maternal-neonatal HIV transmission.

A model of vertical HIV transmission was developed using oral HIV-2(287) exposure of newborn Macaca nemestrina. The minimal Animal Infectious Dose for this oral route was found to be 10-fold higher than that for atraumatic viral transmission across other mucosal membranes (vaginal/rectal) of juvenile macaques. However, once infection was established, viral replication was rapid and plasma viremia could be detected by reverse-transcriptase polymerase chain reaction and viral co-culture within 1 week following exposure. No animal was resistant to infection and all macaques initially exposed to a subinfectious viral inoculum were subsequently infected by re-exposure of mucosal membranes. Higher viral load during primary infection correlated with a more rapid CD4 depletion; however, all HIV-2(287)-infected animals developed CD4 depletion during the observation period. This animal model can now be used to study early viral replication in the presence and absence of anti-retroviral agents to help identify conditions to reduce vertical HIV transmission in human newborns.

Systemic and intestinal immune responses to HIV-2287 infection in Macaca nemestrina.

Nonhuman primate models of human AIDS have been used successfully to evaluate candidate vaccines and infection intervention therapies. Successes of pathogenicity studies in primate models have been limited because of the varied infection outcomes and characteristic low number of study animals. The acutely pathogenic HIV-2(287)--Macaca nemestrina model has shown promise both in antiviral drug evaluation and in pathogenicity studies. Here we describe virus replication, spread, and host responses during the first 28 days of HIV-2(287) infection. Focusing on 18 macaques from a larger 27-macaque study, we report changing virus loads, CD4(+) cell depletions, and antibody responses both systemically and in the mucosa of the small intestine. After intravenous inoculation, blood and intestinal tissue were collected from pairs of macaques at 12 hr and 1, 2, 4, 6, 10, 14, 21, and 28 days postinfection. Specimens were examined for evidence of infection by quantitative cultures, in situ hybridization, lymphocyte subset monitoring, and antibody production. The data were presented serially as though all samples were collected from a single macaque. The highest blood virus loads were detected between days 10 and 14 and subsequently decreased through day 28. This coincided with a significant increase in ileum mucosa virus loads on day 10, which became undetectable by day 28. The lowest levels of CD4(+) cells were observed on days 21 and 28 in blood and ileum mucosa. CD4(+):CD8(+) cell ratios in blood and ileum dropped dramatically after day 10 to lowest levels by day 28. Intestinal virus loads were inversely correlated with CD4(+) cell and virus-specific antibody levels in the ileum after day 6. These results underscore the suitability of this model for pathogenicity studies as well as the importance of the intestinal lymphoid tissues as an initial site of virus replication and cell destruction during the acute, asymptomatic stage of AIDS development.

Characterization of a maternal-fetal HIV transmission model using pregnant macaques infected with HIV-2(287).

To study mechanisms involved in mother-to-fetus transmission of human immunodeficiency virus (HIV) in utero, we have developed a chronically catheterized pregnant macaque model that permits simultaneous and sequential determination of virus in maternal and fetal blood and amniotic fluid during pregnancy. In this report, we have characterized this model using three groups of pregnant macaques designed to sample: (1) maternal blood, fetal blood, and amniotic fluid (n = 6); (2) maternal blood and amniotic fluid (n = 6); or (3) maternal blood only (n = 2). After inoculation with the highly pathogenic HIV-2(287), all pregnant macaques developed brief but intense viremias followed by precipitous CD4+ T-cell declines within 2-3 weeks. While all the infants born to dams of the three groups were HIV positive, the degree of infection and outcome of HIV infection varied. All infants were shown to be HIV-RNA-positive by reverse transcriptase-polymerase chain reaction (RT-PCR). However, HIV-infected cells were detected only in the blood of those born to dams enrolled in groups 1 and 2: most of these infants progressed to CD4+ T-cell depletion. The infants in group 3 exhibited HIV-RNA in plasma, although neither HIV-infected cells nor CD4+ T-cell depletion was detectable. However, all infants developed HIV-2-specific antibody at various levels by 2 months of age. Together, the data suggest that, while the degree of instrumentation may modulate intensity of virus transmission to fetus, the highly pathogenic HIV-2(287) exhibited a high frequency of virus transmission from the mother to fetus.

Suppression of maternal virus load with zidovudine, didanosine, and indinavir combination therapy prevents mother-to-fetus HIV transmission in macaques.

Recently, we developed a maternal-fetal macaque model using a highly pathogenic HIV-2 strain, HIV-2287, to study the time course of HIV transmission in utero. Most pregnant macaques (Macaca nemestrina) infected with HIV-2287 (10-103 infective doses) transmitted HIV to their fetuses, as verified by positive identification of virus-infected mononuclear cells and free viral RNA in fetal blood. To determine whether an antiretroviral drug combination therapy composed of two dideoxynucleosides, azidothymidine (15 mg/kg) and dideoxyinosine (15 mg/kg), and a protease inhibitor, indinavir (25 mg/kg), could completely inhibit mother-to-fetus HIV transmission, we administered these drugs orally through gastric catheters to five pregnant macaques infected with 10 infective doses of HIV-2287. Beginning 30 minutes after HIV inoculation, the dams were given the combination antiviral therapy three times daily until delivery by cesarean section. Drug treatment reduced the maternal virus load to a minimally detectable level but did not prevent primary HIV-2287 infection. All fetal and infant blood samples were virus negative by internally controlled RNA polymerase chain reaction (QC-RNA-PCR) and virus coculture assays. Fetal and infant CD4+ T-cell levels remained normal throughout the experiment. These findings strongly suggest that combination chemotherapy with azidothymidine, dideoxyinosine, and indinavir can suppress maternal viral load enough to prevent mother-to-fetus transmission of HIV.

Protection of Macaca nemestrina from disease following pathogenic simian immunodeficiency virus (SIV) challenge: utilization of SIV nucleocapsid mutant DNA vaccines with and without an SIV protein boost.

Molecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replicating virus. A total of 11 Macaca nemestrina macaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA-immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P = 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4(+) T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with a pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.

Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina).

Non-human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV-1) and animals that are readily available for research. HIV-1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow-up period. These data suggest that a useful non-human primate model for AIDS can be attained in pigtailed macaques (M. nemestrina).

Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina.

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS-like diseases or CD4+ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV-1 infection of humans, including lymphadenopathy, anemia, CD4+ cell depletion, and opportunistic infections. A cell-free virus stock was established from the lymph nodes of an animal that developed AIDS-like diseases. This virus, HIV-2/287, was highly pathogenic in M. nemestrina, causing CD4+ cell depletion within 2-8 weeks postinfection. While both HIV-2 EHO and HIV-2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4 cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.

Enhanced safety and efficacy of live attenuated SIV vaccines by prevaccination with recombinant vaccines.

The only vaccines shown to be protective against intravenous challenge with virulent virus in the simian immunodeficiency virus (SIV)/macaque model are attenuated live SIVs. However, these vaccines have several disadvantages: 1) they persist indefinitely in vaccinated macaques; 2) they are pathogenic to neonatal macaques; and 3) they are lethal in some adult macaques. To enhance the safety and efficacy of these vaccines, we immunized macaques first with recombinant vaccines and then inoculated the animals with SIV(delta(nef)). In the first experiment, preimmunized macaques advanced to disease slower than controls after challenge with virulent SIV; five animals survived for 3 years without disease and only the vaccine virus (SIV(delta(nef)) could be isolated at this time. In the second experiment, preimmunized animals had lower virus loads and no disease compared to controls.

Post-exposure chemoprophylaxis (PECP) against SIV infection of macaques as a model for protection from HIV infection.

We report that simian immunodeficiency virus (SIV) infection in macaques is a valuable animal model for studying post-exposure chemoprophylaxis (PECP). PECP with the acyclic nucleoside reverse transcriptase inhibitors 9-(2-phosphonylmetho-xyethyl)adenine (PMEA) and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) at early viral infection can provide long-term protection against subsequent heterologous SIV challenge. Eight macaques previously treated with PECP (called PECP macaques) and four naive controls were challenged intravenously with the most virulent form of SIV, SIV(PBj14). All controls showed signs of SIV(PBj14)-induced acute disease syndrome on days 6 and 7 post-inoculation (PI). One had a fatal viral infection and two surviving controls had persistent infection and decreased CD4+ cell count. Virologic studies of the three surviving controls revealed SIV in multiple lymphoid tissues and peripheral blood mononuclear cells (PBMCs) at necropsy. In contrast, the PECP macaques showed none to mild signs of acute disease syndrome at day 9 PI and exhibited only transient SIV infection in PBMCs between weeks 1 and 8 PI. In virologic studies of five PECP macaques necropsied, two macaques were SIV-negative and the other three were SIV-positive only in either lymph node or bone marrow. Three SIV(PBj14)-challenged PECP macaques, that were randomly reserved for a follow-up study for > 4.0 years PI showed extremely low to undetectable levels of PBMC-associated viremia and normal to increased levels of CD4 + and CD8 + cell counts throughout the study. Our results indicate that early PECP could activate immune responses to protect against subsequent infection with heterologous challenge virus.

Rapid shift from virally infected cells to germinal center-retained virus after HIV-2 infection of macaques.

Lymphoid tissues are the primary target during the initial virus dissemination that occurs in HIV-1-infected individuals. Recent advances in antiretroviral therapy and techniques to monitor virus load in humans have demonstrated that the early stages of viral infection and host response are major determinants of the outcome of individual infections. Relatively little is known about immunopathogenic events occurring during the acute phase of HIV infection. We analyzed viral dissemination within lymphoid tissues by in situ hybridization and by combined immunohistochemistry/in situ hybridization during the acute infection phase (12 hours to 28 days) in pig-tailed macaques (Macaca nemestrina), challenged intravenously with a virulent strain of HIV-2, HIV-2(287). Two stages in viral dissemination were clearly evident within the first 28 days after HIV-2(287) infection. First, a massive increase in individual HIV-2-infected cells, mostly CD3+ T lymphocytes and a smaller percentage of macrophages and interdigitating dendritic cells, was identified within lymph nodes which peaked on the 10th day after HIV-2 infection. A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection. Coincident with a marked reduction in individual HIV-2 RNA+ cells by day 14 postinfection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection. Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells. In this nonhuman primate model of HIV-associated immunopathogenesis, using a virus derived from a human pathogen, we identified a significant shift in the pattern of HIV-2 localization within a narrow time frame (day 10 to day 14). This shift in virus localization and behavior indicates that there may be a discrete but remarkably narrow window for therapeutic interventions that interrupt this stage in the natural course of HIV infection. Reproducibility and the accelerated time course of disease development make this model an excellent candidate for such intervention studies.

Antigen-specific cytokine responses in vaccinated Macaca nemestrina.

We describe a new surrogate assay for CD8 + T lymphocyte activity that has the capability of discriminating between cytotoxic T lymphocyte (CTL) activity and cytokine-mediated suppressive activity. We applied this approach to two groups of Macaca nemestrina vaccinated with a minimally pathogenic strain of human immunodeficiency virus type 2 [HIV-2 (HIV-2(KR))] as a model of an attenuated virus vaccine. Group 1 was then inoculated with a non-infectious stock of a pathogenic strain, HIV-2287. Both groups 1 and 2 were subsequently challenged with an infectious stock of HIV-2287. Five out of six group 1 animals were protected against CD4 decline, whereas three out of six animals in group 2 were protected. Analysis of CTL responses demonstrated strong activity against HIV-2(KR)-Gag in group 1. It was determined that strong CTL responses correlate with antigen-specific T-helper (Th) type 1 responses. This antigen-specific cytokine assay has the potential to better elucidate the functional mechanisms of CD8 + T-cell-mediated protection than traditional methods to date.

Immunization against SIVmne in macaques using multigenic DNA vaccines.

All structural and regulatory genes of SIVmne were cloned into mammalian expression vectors to optimize expression in vitro and immunogenicity in mice. Macaca fascicularis were immunized four times with plasmid DNA (n = 4), or two DNA priming inoculations followed by two boosts of recombinant gp160 plus Gag-Pol particles (n = 4). Following intrarectal challenge with SIVmne, all macaques became infected. Three monkeys immunized with DNA alone maintained low plasma virus loads by 1 year post-challenge; the fourth exhibited high virus loads and significant CD4+ cell decline. Two of the DNA plus boost and three control macaques had high virus loads and associated CD4+ cell decline. Both vaccine protocols elicited antibodies and comparable helper T-cell proliferative responses to gp160. Cytokine mRNA levels in activated peripheral blood mononuclear cells (PBMC) taken at time of challenge suggested a dominant T helper (Th) 1 state in three DNA-immunized and one protein-boosted macaque, which correlated with low virus loads and high CD4+ cell counts post-challenge.

Role of immune responses against the envelope and the core antigens of simian immunodeficiency virus SIVmne in protection against homologous cloned and uncloned virus challenge in Macaques.

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.

Thrombotic microangiopathy in the HIV-2-infected macaque.

Thrombotic microangiopathy (TMA) has been increasingly reported in human immunodeficiency virus (HIV)-infected humans over the past decade. The pathogenesis is unknown. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques (Macaca nemestrina), infected intravenously with a virulent HIV-2 strain, HIV-2(287), in addition to that of four uninfected control macaques. Necropsies were performed between 12 hours and 28 days after infection. HIV-2 antigen was detectable in peripheral blood mononuclear cell (PBMC) cocultures in all animals after 10 days of HIV-2 infection; a rapid decline in CD4(+) PBMC (<350/microliter) was seen in five of six animals 21 days and 28 days after infection. No macaque developed features of clinical AIDS. Typical lesions of human HIV-associated nephropathy were undetectable. Six of the 27 HIV-2-infected macaques demonstrated both histological TMA lesions (thrombi in glomerular capillary loops and small arteries, mesangiolysis) and ultrastructural lesions (mesangiolysis, subendothelial lucency, platelet thrombi in glomerular capillary lumina). Extrarenal thrombi were detected in the gastrointestinal and adrenal microvasculature of macaques that had developed renal TMA. None of the control animals demonstrated features of renal TMA at necropsy. In a retrospective analysis of kidneys obtained from 39 additional macaques infected with HIV-2(287), seven cases demonstrated TMA. In situ hybridization showed no detectable HIV-2 RNA in kidney sections of 65/66 HIV-2-infected macaques, including all 13 TMA cases. Expression of the chemokine receptor CXCR4, the putative coreceptor for HIV-2(287), was absent in intrinsic renal cells in all HIV-2-infected macaques. The HIV-2-infected macaque may be a useful model of human HIV-associated TMA. Our data do not support a role of direct HIV-2 infection of intrinsic renal cells as an underlying mechanism.

Protection from pathogenic SIV challenge using multigenic DNA vaccines.

To assess DNA immunization as a strategy for protecting against HIV infection in humans, we utilized SIVmne infection of Macaca fascicularis as a vaccine challenge model with moderate pathogenic potential. We compared the efficacy of DNA immunization alone and in combination with subunit protein boosts. All of the structural and regulatory genes of SIVmne clone 8 were cloned into mammalian expression vectors under the control of the CMV IE-1 promoter. Eight M. fascicularis were immunized twice with 3 mg of plasmid DNA divided between two sites; intramuscular and intradermal. Four primed macaques received a further two DNA immunizations at weeks 16-36, while the second group of four were boosted with 250 microg recombinant gp160 plus 250 microg recombinant Gag-Pol particles formulated in MF-59 adjuvant. Half of the controls received four immunizations of vector DNA; half received two vector DNA and two adjuvant immunizations. As expected, humoral immune responses were stronger in the macaques receiving subunit boosts, but responses were sustained in both groups. Significant neutralizing antibody titers to SIVmne were detected in one of the subunit-boosted animals and in none of the DNA-only animals prior to challenge. T-cell proliferative responses to gp160 and to Gag were detected in all immunized animals after three immunizations, and these responses increased after four immunizations. Cytokine profiles in PHA-stimulated PBMC taken on the day of challenge showed trends toward Thl responses in 2/4 macaques in the DNA vaccinated group and in 1/4 of the DNA plus subunit vaccinated macaques; Th2 responses in 3/4 DNA plus subunit-immunized macaques; and Th0 responses in 4/4 controls. In bulk CTL culture, SIV specific lysis was low or undetectable, even after four immunizations. However, stable SIV Gag-Pol- and env-specific T-cell clones (CD3+ CD8+) were isolated after only two DNA immunizations, and Gag-Pol- and Nef-specific CTL lines were isolated on the day of challenge. All animals were challenged at week 38 with SIVmne uncloned stock by the intrarectal route. Based on antibody anamnestic responses (western, ELISA, and neutralizing antibodies) and virus detection methods (co-culture of PBMC and LNMC, nested set PCR- of DNA from PBMC and LNMC, and plasma QC-PCR), there were major differences between the groups in the challenge outcome. Surprisingly, sustained low virus loads were observed only in the DNA group, suggesting that four immunizations with DNA only elicited more effective immune responses than two DNA primes combined with two protein boosts. Multigenic DNA vaccines such as these, bearing all structural and regulatory genes, show significant promise and may be a safe alternative to live-attenuated vaccines.

Protection of macaques against intrarectal infection by a combination immunization regimen with recombinant simian immunodeficiency virus SIVmne gp160 vaccines.

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.

Limited breadth of the protective immunity elicited by simian immunodeficiency virus SIVmne gp160 vaccines in a combination immunization regimen.

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.

Chemokine receptor (CCR5) expression in human kidneys and in the HIV infected macaque.

BACKGROUND: The chemokine receptor, CCR5, has been identified as an essential co-receptor with CD4, which permits entry of human immunodeficiency virus (HIV) into mammalian cells. This receptor may also mediate leukocyte and parenchymal responses to injury by virtue of its binding to locally released chemokines such as RANTES, MIP-1 alpha and MIP-1 beta during inflammation. The localization of CCR5 in human or primate kidney is unknown. In this study we sought to identify sites of CCR5 synthesis through localization of mRNA coding for this peptide. METHODS: CCR5 cDNA cloned into an expression vector was transcribed into a 1.1 Kb antisense riboprobe that was utilized for in situ hybridization (ISH) and Northern blotting studies. RESULTS: Northern analysis demonstrated positive hybridization for CCR5 mRNA in total RNA isolated from allograft nephrectomy tissue with features of severe transplant rejection as well as in kidney tissue with focal interstitial nephritis. No comparable hybridization signal was achieved with human kidney tissue uninvolved by disease. CCR5 mRNA was not identified in intrinsic renal cell types by ISH in normal human (N = 6), normal macaque kidney (N = 5), in kidneys from macaques with established infection by HIV-2 (N = 9), kidneys from macaques infected with HIV-1 (N = 4), nor in kidneys from SIV-infected macaques (N = 5). CCR5 was identified by ISH in human kidneys with features of interstitial nephritis (N = 3) and in rejected human allograft kidneys (N = 14). The expression of CCR5 was restricted to infiltrating mononuclear leukocytes at sites of chronic tubulointerstitial injury and at sites of vascular and interestitial rejection, respectively. CONCLUSIONS: Understanding the localization of CCR5 as well as other chemokine receptors may help us understand how specificity in leukocyte trafficking is achieved in renal inflammatory processes such as allograft rejection and interstitial nephritis. They provide additional evidence that chemokines may be critical mediators of leukocyte trafficking in renal allograft rejection. These findings may account in part for the difficulty in demonstrating HIV infection of renal cells in human HIV infection, since these cells appear to lack constitutive expression of an essential co-receptor needed for viral entry.

Mucosal antibody expression following rapid SIV(Mne) dissemination in intrarectally infected Macaca nemestrina.

The early kinetics of antibody expression following transmucosal infection by SIV(Mne) were examined in several mucosal compartments in Macaca nemestrina. Five male-female pairs of macaques were inoculated intrarectally with SIV(Mne) E11S, a biological clone, and serially euthanized at 1, 2, 4, 8, and 12 weeks postinoculation. Plasma, tears, saliva, rectal secretions, and vaginal washes were collected serially and just prior to euthanasia. Both total and SIV-specific IgG and IgA levels were measured by immunoglobulin isotype-specific quantitative enzyme-linked immunosorbent assays (ELISAs), and were further examined by conventional and enhanced chemiluminescence (ECL) immunoblots. Virus coculture, polymerase chain reaction, and in situ hybridization assays revealed the systemic spread of virus as early as 1 week postinoculation in 8 of 10 animals. ECL immunoblots detected SIV-specific antibodies in mucosal samples collected 1 week postinoculation. The most dramatic increases in both total and SIV-specific IgA levels were detected in rectal secretion samples. In contrast, plasma and nonrectal mucosal samples from the same time points increased only slightly, suggesting that the most robust antibody response occurred at the portal of infection. Our results show that the SIV-infected macaque is an excellent model for studies designed to assess mucosal immune responses to primate lentivirus infections. Additional studies will assess the correlation between the antiviral protection afforded by candidate vaccines and mucosal antibody responses.

Mechanisms of clearance of Treponema pallidum from the CSF in a nonhuman primate model.

OBJECTIVES: To establish a model of CNS invasion by Treponema pallidum and to use it to investigate the immune mechanisms responsible for clearance. METHODS: Four macaques were intrathecally inoculated with 0.6 to 2.1 x 10(8) T. pallidum and underwent clinical examinations and blood and CSF collections every 1 to 2 weeks for 12 to 13 weeks. The following were determined: serum Venereal Disease Research Laboratory (VDRL) and microhemagglutination-T. pallidum reactivities, CSF-VDRL, CSF white blood cell (WBC) count, and the presence of viable T. pallidum in CSF by the rabbit infectivity test (all animals), as well as the presence of T. pallidum in CSF by reverse transcriptase (RT)-PCR, WBC phenotype by fluorescence-activated cell sorter, WBC cytokine production by RT-PCR, and brain MRI at 10 weeks (two animals). RESULTS: All animals became systemically infected and developed CSF pleocytosis that resolved after 8 weeks. CSF T. pallidum was detected from 2 to 8 weeks. CSF T lymphocytes were predominantly CD4+. Interferon-gamma (IFN-gamma) mRNA was consistently detected in CSF WBCs, but interleukin (IL)-4 and IL-5 were not. All animals remained clinically well. MRIs were normal. CONCLUSIONS: In this model, T. pallidum is cleared from the CNS just as in most humans with early syphilis. Local production of IFN-gamma likely participates in this process. This model could be used to clarify the effect of retrovirus-induced immunodeficiency on clearance of T. pallidum from the CNS and on the local CNS immune response.