Protection of macaques against SIV infection by subunit vaccines of SIV envelope glycoprotein gp160.

Simian immunodeficiency virus (SIV) is a primate lentivirus related to human immunodeficiency viruses and is an etiologic agent for acquired immunodeficiency syndrome (AIDS)-like diseases in macaques. To date, only inactivated whole virus vaccines have been shown to protect macaques against SIV infection. Protective immunity was elicited by recombinant subunit vaccines. Four Macaca fascicularis were immunized with recombinant vaccinia virus expressing SIVmne gp160 and were boosted with gp160 produced in baculovirus-infected cells. All four animals were protected against an intravenous challenge of the homologous virus at one to nine animal-infectious doses. These results indicate that immunization with viral envelope antigens alone is sufficient to elicit protective immunity against a primate immunodeficiency virus. The combination immunization regimen, similar to one now being evaluated in humans as candidate human immunodeficiency virus (HIV)-1 vaccines, appears to be an effective way to elicit such immune responses.

Infection of Macaca nemestrina by human immunodeficiency virus type-1.

After observations that Macaca nemestrina were exceptionally susceptible to simian immunodeficiency virus and human immunodeficiency virus type-2 (HIV-2), studies of HIV-1 replication were initiated. Several strains of HIV-1, including a recent patient isolate, replicated in vitro in peripheral blood mononuclear cells (PBMCs) and in CD4-positive M. nemestrina lymphocytes in a CD4-dependent fashion. Eight animals were subsequently inoculated with either cell-associated or cell-free suspensions of HIV-1. All animals had HIV-1 isolated by cocultivation, had HIV-1 DNA in their PBMCs as shown by polymerase chain reaction, and experienced sustained seroconversion to a broad spectrum of HIV-1 proteins. Macaca nemestrina is an animal model of HIV-1 infections that provides opportunities for evaluating the pathogenesis of acute HIV-1 replication and candidate vaccines and therapies.

Characterization of exogenous type D retrovirus from a fibroma of a macaque with simian AIDS and fibromatosis.

A novel type D retrovirus was isolated by cocultivation of explants of fibromatous tissue from a rhesus monkey (Macaca mulatta) with immunodeficiency and retroperitoneal fibromatosis. This type D virus, isolated from a macaque with simian acquired immunodeficiency syndrome (SAIDS-D/Washington), is exogenous and is partially related to the Mason-Pfizer and the langur monkey type D viruses. The SAiDS-D virus can be distinguished from all other primate retroviruses by antigenicity and molecular hybridization. Nucleic acid hybridization studies reveal that the origin of the SAIDS-D isolate may reside in Old World monkey (subfamily Colobinae) cellular DNA.

Cytotoxic and proliferative T cell responses in HIV-1-infected Macaca nemestrina.

Macaca nemestrina has been described as an animal model for acute HIV-1 infection. This animal, unlike most infected humans, appears to contain HIV-1 replication. Therefore analysis of HIV-1-specific proliferative and cytotoxic T lymphocyte (CTL) responses following HIV-1 challenge of M. nemestrina may provide information into the role of such responses in both the control of acute HIV infection and protective immunity. Although CD4+ T cell responses to HIV-1 are generally difficult to detect in HIV-1-infected humans, early and persistent CD4+ T cell proliferative responses to HIV-1 antigens were detected in all HIV-1-inoculated M. nemestrina. HIV-1-specific CD8+ CTL responses were evaluated in PBMC by stimulation with autologous cells expressing HIV-1 genes, limiting dilution precursor frequency analysis, and T cell cloning. CTL reactive with gag, env, and nef were present 4-8 wk after infection, and persisted to 140 wk after infection. The presence of both CD4+ and CD8+ T cell responses before and after clearance of HIV-1 viremia is consistent with a role for these responses in the successful control of HIV-1 viral replication observed in M. nemestrina. Further studies of T cell immunity in these animals that resist disease should provide insights into the immunobiology of HIV-1 infection.

Infection of Macaca nemestrina neonates with HIV-1 via different routes of inoculation.

OBJECTIVES: Receptive anal intercourse but not orogenital sex has been identified as a major risk factor for transmission of HIV-1. Recent studies using simian immunodeficiency virus (SIV) in rhesus macaques have demonstrated relatively efficient infection following oral administration, indicating that modes of transmission may vary between HIV-1 and SIV. Here, we investigate whether HIV-1 infection of macaques via the oral route is more efficient than via the rectal route. DESIGN: Eleven Macaca nemestrina neonates were exposed to HIV-1 via different routes (four oral, two intravenous, and five rectal). One animal was orally inoculated with a sham inoculum and two control animals were not exposed. METHODS: All animals were followed for virological signs of infection, and for pathogenesis associated with HIV-1 infection by general physical examinations, complete blood cell counts and lymphocyte subset analysis, and full necropsies. RESULTS: Three out of five rectally exposed macaques and both of the intravenously inoculated animals became infected with HIV-1, whereas none of the orally exposed animals showed evidence of HIV-1 infection. Clinical observations following exposure included failure to thrive in the orally inoculated animals and low CD4/CD8 ratios in the rectally exposed macaques. CONCLUSIONS: The finding that, contrary to what has been reported for SIV, transmission of HIV-1 via the oral route is not more efficient than via the rectal route, indicates important biological differences between HIV-1 and SIV, with direct implications for the spread of HIV and associated AIDS, and for development of anti-HIV-1 vaccines.

T-cell proliferation to subinfectious SIV correlates with lack of infection after challenge of macaques.

OBJECTIVES: To analyze correlates of protection in macaques exposed to SIV. METHODS: Peripheral blood mononuclear cells (PBMC) from macaques inoculated intrarectally with various dilutions of SIV were examined for their in vitro proliferative response to SIV envelope peptides and generation of SIV-specific antibodies. Some macaques previously exposed intravenously to subinfectious doses of SIV were subsequently challenged 16 months later with an infectious intrarectal dose of SIV. RESULTS: The viral-specific immune responses of macaques exposed to infectious doses of SIV were characterized by generation of antibodies and weak or undetectable T-cell-mediated responses. In contrast, macaques inoculated with doses of SIV below the threshold required for seroconversion and recovery of virus exhibited T-cell proliferation in response to SIV envelope synthetic peptides. The macaques that had previously been exposed to SIV resisted the subsequent virus challenge, whereas the naive macaques (never exposed to SIV) all became infected. CONCLUSIONS: The inability to productively infect macaques previously exposed to subinfectious doses of SIV suggests that a T-cell-mediated response may confer long-term protection against infection, and that AIDS vaccines should be designed to optimize the cellular arm of the immune response.

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.

Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection.

Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIVmne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIVmne. Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp 130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core (gag-pol) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10-100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorporate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).

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.

Antibody recognition of SIVmac envelope peptides in plasma from macaques experimentally infected with SIV/Mne.

Four stretches of amino acid sequences encoded in conserved HIV-1 env domains and four parallel regions of the SIVmac env (two from gp120 and two from gp41/p32E) were fused to the NH2 terminus of beta-galactosidase by recombinant DNA techniques and used to analyze sera from three macaque species experimentally infected with SIV/Mne. All SIVmac env sequences were recognized by sera from the SIV/Mne-inoculated macaques. Western blot analysis performed with whole SIV/Mne, SIVmac, SIVagm, and HIV-1 antigens and sera from SIV/Mne-infected macaques also demonstrates that SIV/Mne is immunologically more closely related to SIVmac than to SIVagm or to HIV-1. Antibody levels to the gp120 NH2-terminal SIV-88 epitope appear to decrease in the infected Macaca nemestrina with progression of disease, as was also reported for the parallel HIV-1 epitope in HIV-1-infected individuals. Sera from all infected macaques reacted with the p32E-SIV-582 epitope (EKYLEDQAQLNAWGCAFRQVC). High titers to this immunodominant epitope could be detected at least 9 weeks postinfection and at a time when primarily the p28 and p32E antibodies were detectable in Western blots performed with whole disrupted SIV/Mne virus. In the majority of animals, antibody titers of 1:100,000 to SIV-582 develop during the infection and persist until death. Antibody responses to the SIV env epitopes in SIV/Mne-infected macaques thus resemble in many aspects (prevalence and immunogenicity) those observed previously for the corresponding HIV-1 env epitopes in HIV-1-infected humans.

Simian immunodeficiency virus infection of macaque primary placental cells.

We have characterized the ability of a simian immunodeficiency virus, SIVmne strain E11S, to infect macaque placental trophoblast and Hofbauer cells. These primary placental cells were permissive to SIVmne infection, regardless of gestational age. Virus production by the infected cells was determined as time-dependent viral core antigen p27 production, followed by verification of the proviral gag/LTR DNA sequences in the infected cells using a polymerase chain reaction assay. Of more than six placentas tested, SIVmne infection of placental cells at an early gestational age (i.e., days 55 or 78) produced more than 10-fold the amount of virus core antigen p27 than did placental cells infected at a late gestational age (i.e., days 135 or 165). In addition, SIVmne infection of trophoblast cells was inhibited by SIVmac neutralizing macaque serum but not by normal serum, indicating the specificity of virus infection. Furthermore, the amount of SIV core antigen p27 produced by the virus-infected trophoblast and Hofbauer cells was shown to be dependent on the multiplicity of virus infection. Collectively, our results indicate that macaque trophoblast and Hofbauer cells can be infected by SIV and that both gestational age and viral dose may play a role in the extent of viral infection.

Analysis of cytotoxic T lymphocyte responses to SIV proteins in SIV-infected macaques using antigen-specific stimulation with recombinant vaccinia and fowl poxviruses.

Methods to analyze CD8+ CTL responses to simian immunodeficiency virus (SIV)-encoded proteins are essential to understand lentivirus immunopathogenesis and protective immune responses. Recombinant infectious shuttle vectors are useful for analyzing CTL responses to many viruses, including HIV. Therefore, CTL responses in SIV-infected Macaca fascicularis to SIV env and SIV gag/pol were evaluated using specific antigen stimulation with recombinant vaccinia (rVV) and fowl poxviruses (rFPV) containing SIV genes. Peripheral blood mononuclear cells from SIV-infected animals were stimulated with autologous cells infected with rVV expressing SIV env/gag/pol, and CTLs specific for SIV env and for SIV gag/pol were detected by testing for lytic activity in target cells expressing these genes separately. Lymphocyte subset purifications from the effector population demonstrated that the CTL response was mediated by CD8+ cells, and the use of brefeldin A to selectively block antigen presentation in association with MHC class I products affirmed this cytolytic activity was class I restricted. The use of rVV to analyze responses to SIV genes is potentially problematic in hosts immunized to vaccinia. Fowl poxvirus is an alternative virus that has many of the molecular advantages of vaccinia virus but is genomically distinct. Therefore, the ability of rFPV to expand and detect SIV-specific CTLs was evaluated. Although there was no cytopathic effect following infection with rFPV, macaque cells infected with this vector did express rFPV gene products, and could be used as stimulator and target cells to detect SIV-specific CD8+ CTLs. The results suggest that these recombinant viral vectors can be used to specifically stimulate CD8+, MHC class I-restricted CTLs reactive to SIV proteins, and should facilitate evaluating CTL responses in both SIV-infected animals and animals vaccinated against SIV.

Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques.

Reports of significant reductions in plasma viral load by anti-HIV drugs have raised the possibility that antiviral therapy, if initiated sufficiently early, may result in sustained control of infection and prolonged clinical benefits. We evaluated the effects of intervention coincident with infection using an antiviral nucleoside, d4T, in Macaca nemestrina infected with a highly pathogenic isolate of HIV-2 (HIV-2[287]). Infection with this virus reproducibly results in high viremia and rapid CD4+ cell depletion, allowing a sensitive measurement of the treatment effect on viral load and clinical outcome. Compared to the control group, d4T-treated macaques showed significantly lower (2-3 log10) plasma- and cell-associated viral load. No CD4+ cell decline was observed in the treatment group while on therapy with d4T whereas CD4+ cells of control macaques declined from a preinfection mean of 32% of PBMCs to below 10%. Notably, when d4T treatment was withdrawn after 16 weeks, five of the six macaques continued to control viral load and have maintained normal CD4+ cell level for more than a year. These results demonstrate that early antiviral intervention, even of a limited duration, may constitute an important strategy against lentiviral-induced disease.

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.

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.

Focal segmental glomerulosclerosis in primates infected with a simian immunodeficiency virus.

Focal and segmental glomerulosclerosis (FSG) with endothelial tubuloreticular inclusions (TRIs) is the typical lesion of human HIV-associated glomerulopathy. Autopsy studies showed the presence of FSG in 3 of 15 macaques dying 15-120 weeks after experimental infection with a simian immunodeficiency virus (SIVMne). Ultrastructural studies generally revealed numerous endothelial TRIs (also present in normals), mesangial expansion, and evidence of mesangial cell injury. One additional animal had a small-vessel polyarteritis with a proliferative and focally crescentic glomerulonephritis; seven animals had mild, multifocal interstitial nephritis. All animals had documented viremia after infection; 14 of 15 developed antibodies to SIV postinoculation. Additional postmortem findings included severe enterocolitis, encephalitis, and opportunistic infections. In contrast, autopsy studies of macaques infected with a type D simian retrovirus (SAIDS-D/Washington, SRV-2) for similar periods of time (n = 40) showed no evidence of FSG. One SRV-infected animal had a mild proliferative glomerulonephritis. These studies indicate SIV-infected primates may provide a relevant model for study of human HIV-associated nephropathy. They also indicate the variable pathology that can be seen in primate infections of distinct retrovirus types, each of which produces a simian immunodeficiency state that resembles human AIDS.

Biosynthesis of fatty acids from acetate in soybean suspension cultures.

Suspension cultures of finely divided soybean cells established from callus were incubated with sodium [1(14)C] acetate for periods up to 86 hr. Lipids and fatty acids were analyzed for radioactivity in samples harvested at logarithmic time periods. Incorporation of acetate into cell lipid was directly proportional to the logarithm of time up to 32 hr, after an initial lag of 4-6 hr. Most of the lipid radioactivity was found in the phospholipid fraction, and all common soybean fatty acids became labeled within 6 hr. The order of labeling and distribution of radioactivity with time were essentially the same as in tissues from intact growing plants. These results support the concept of sequential desaturation of oleic acid in the cells. It was concluded that valid studies of the biosynthesis of common lipids in the soybean can be carried out for extended periods of time by use of undifferentiated cells in suspension cultures.