Resistance of neonatal monkeys to live attenuated vaccine strains of simian immunodeficiency virus.

A major safety concern of using live-attenuated vaccine strategies against AIDS is the potential exposure of neonates or fetuses to vaccine virus from the mother. Here we report that high viral loads and disease were observed in only 2 of 18 neonatal monkeys infected with gene-deleted vaccine strains of simian immunodeficiency virus. Pathogenicity was restricted to neonates born to unvaccinated mothers, that is, lacking maternal immunity, and that received extremely high doses of vaccine virus orally. No in utero transmission of vaccine virus was observed in four neonates born to mothers vaccinated during the second trimester. Our results suggest that the live attenuated vaccine approach should remain a viable option for preventing HIV infection and disease in high-risk human populations.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Induction of immune responses and safety profiles in rhesus macaques immunized with a DNA vaccine expressing human prostate specific antigen.

Prostate specific antigen (PSA) is a widely used marker for prostate cancer, which is secreted by normal prostate cells at low levels, but is produced more substantially by cancer cells. We have previously reported on the use of a DNA vaccine construct that encodes for human PSA gene to elicit host immune responses against cells producing PSA. DNA immunization strategy delivers DNA constructs encoding for a specific immunogen into the host, who becomes the in vivo protein source for the production of antigen. This antigen then is the focus of the resulting immune response. In this study, we examine the induction of immune responses and safety profiles in rhesus macaques immunized with DNA-based PSA vaccine. We observed induction of PSA-specific humoral response as well as positive PSA-specific lymphoproliferative (LPA) response in the vaccinated macaques. We also observed that the stimulated T cells from the PSA-immunized rhesus macaques produced higher levels of Th1 type cytokine IFN-gamma than the control vector immunized animals. On the other hand, DNA immunization did not result in any adverse effects in the immunized macaques, as indicated by complete blood counts, leukocyte differentials and hepatic and renal chemistries. The macaques appeared healthy, without any physical signs of toxicity throughout the observation period. In addition, we did not observe any adverse effect on the vaccination site. The apparent safety and immunogenecity of DNA immunization in this study suggest that further evaluation of this vaccination strategy is warranted.

Simian immunodeficiency virus infection via amniotic fluid: a model to study fetal immunopathogenesis and prophylaxis.

The rising prevalence of infection with the human immunodeficiency virus type 1 (HIV-1) in young women will increase the number of infected children worldwide. Because HIV-1 seems to be transmitted mostly intrapartum, fetal infection probably occurs mainly via skin or mucous membrane exposure. A model for this route of fetal infection has been established in primates. After injecting the simian immunodeficiency virus (SIV) into amniotic fluid during late gestation, six of seven rhesus monkeys were born infected. All infected neonates were viable and showed signs of disease, such as low birth weights, lymphadenopathy, and rashes. Cytotoxic T-cell responses to SIV were absent in neonates, but present in mothers. The high fetal infection rate allows studies of lentiviral immunopathogenesis during ontogeny and the development of strategies to prevent maternal HIV-1 transmission.

Use of particulate forms of protein antigens to increase the sensitivity of antigen-specific proliferation assays.

Different forms of recombinant HIV-1 gp160 and tetanus toxoid were adsorbed onto latex microspheres and this particulate form of the proteins was used to measure antigen-specific proliferation in vaccinated rhesus macaques. Proliferative responses to proteins bound to microspheres were significantly greater and allowed for the detection of antigen-specific responses that were not detected using soluble proteins. The responses were antigen-specific and required prior immunization of the animals. Additionally, the presence and magnitude of the proliferative responses was associated with antibody responses to the same proteins suggesting the results were representative of in vivo responses and that the assay format did not induce in vitro artifacts.

The use of SIV-infected rhesus monkeys for the preclinical evaluation of AIDS drugs and vaccines.

Macaque monkeys infected with the simian immunodeficiency virus (SIV) can be used for preclinical testing of drugs and vaccines against acquired immunodeficiency syndrome (AIDS) as well as for the study of AIDS pathogenesis. A number of pathogenic SIV strains that have been well characterized molecularly and biologically are available for animal infection studies. Data generated from in vitro drug sensitivity assays have established, for many classes of compounds, a similar degree of antiviral efficacy against both HIV-1 and the SIVs, although some examples of selective inhibitors of HIV-1 now are known. A number of virus and host parameters have been defined that provide suitable biological endpoints for in vivo efficacy studies during acute and chronic infection of macaque monkeys. Vaccine studies in SIV-infected monkeys have provided hope that immune protection against lentiviruses is possible; SIV systems are playing a major role in systematically comparing various vaccine strategies to determine correlates of immunity and the protection required for mucosal versus parenteral routes of infection. Societal pressures and the expanding AIDS epidemic will continue to encourage early testing of experimental drugs and vaccines in human clinical trials, however, as more data validating the SIV system are generated, the utility of the SIV model in preclinical development likely will become apparent. Impetus to evaluate therapies in this model system will increase if the current method of testing in humans does not identify more effective AIDS therapies in the near future.

Immunogenicity and toxicity testing of an experimental HIV-1 vaccine in nonhuman primates.

A highly purified saponin from Q. saponaria (QS-21) was tested in juvenile rhesus macaques for adjuvant activity and toxicity. The QS-21 was tested alone or as part of an experimental subunit HIV-1 vaccine containing a truncated recombinant HIV-1 envelope protein (gp160D) adsorbed to alum. Antibody responses were measured using ELISA and cell-mediated immunity was measured using cellular proliferation assays. Potential toxicity was monitored by standard clinical pathology testing using peripheral blood and urine samples. No toxic effects were observed, even after the administration of the experimental vaccines three times at monthly intervals. The QS-21 saponin adjuvant enhanced total antibody production levels by greater than 100-fold and broadened the specificity of the response so that additional epitopes were recognized, when compared with alum-adsorbed HIV-1 gp160D formulation. Low-level, antigen-specific proliferative responses to HIV-1 recombinant gp160 were induced by either vaccine formulation. Proliferative responses were induced by a sham challenge with soluble recombinant HIV-1 gp160 for all of the animals that had been vaccinated. However, those that received the HIV-complete vaccine formulation containing QS-21 responded significantly better. These data demonstrated that the QS-21 adjuvant augmented both antibody responses and cell-mediated immunity and established immunological memory. The potent adjuvant activity and lack of toxicity suggest that this adjuvant should be safe and effective for use in HIV-1 vaccines.

Induction of antigen-specific killer T lymphocyte responses using subunit SIVmac251 gag and env vaccines containing QS-21 saponin adjuvant.

Subunit vaccines based on recombinant proteins have proved useful for inducing antibody responses and they are safe for widespread use because they do not contain any live components. Unfortunately, they do not typically induce the types of cell-mediated immune responses required to control viral pathogens; specifically, they do not induce CD8+ cytotoxic T lymphocyte (CTL) responses. To increase the immunogenicity of recombinant proteins, we have used the QS-21 saponin adjuvant in subunit vaccine formulations. In the current study, experimental subunit vaccine formulations containing recombinant p55gag or gp120env proteins from the mac251 strain of the simian immunodeficiency virus (SIVmac251) and the QS-21 adjuvant were used to immunize rhesus macaques. These formulations induced SIV gag- or env-specific cellular immunity that was detectable in vitro and included killer cell activity. The induction of killer cells required prior vaccination and the responses were antigen specific for the immunogens contained in the vaccine formulations. Autologous target cells were required to detect these responses, suggesting genetic restriction, and effector cells appeared to be present in both the CD4+ and CD8+ T lymphocyte subpopulations. These data suggest that the vaccine-induced killer cell activity that was detected was mediated by both CD4+ and CD8+ lymphocytes. Despite the presence of these killer cells, all of the animals became infected with the SIVmac251 on experimental challenge. These findings demonstrated that antigen-specific killer cell responses could be induced by a subunit vaccine formulated with the QS-21 saponin adjuvant. The characteristics of the responses suggested that the effector cells were T lymphocytes, expressing either CD4 or CD8.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a cellulose acetate phthalate topical cream on vaginal transmission of simian immunodeficiency virus in rhesus monkeys.

Human immunodeficiency virus type 1 (HIV-1) infection continues to spread in developing countries, mostly through heterosexual transmission. The development of a safe and cost-effective topical microbicide, effective against a range of STDs including HIV-1, would greatly impact the ongoing epidemic. When formulated in a vehicle, a micronized form of cellulose acetate phthalate (CAP), which is an inactive pharmaceutical excipient, has been shown to inactivate HIV-1, herpes simplex virus types 1 and 2, cytomegalovirus, Neisseria gonorrhoeae, Trichomonas vaginalis, Haemophilus ducreyi, and Chlamydia trachomatis in vitro. Formulated CAP was also shown to be effective against herpes simplex virus type 2 in vivo. Here we show that a formulation of CAP protected four of six rhesus monkeys from vaginal infection with simian immunodeficiency virus. Thus, CAP may be a candidate for use as a topical microbicide for preventing HIV-1 infection in humans.

Vaccine protection by a triple deletion mutant of simian immunodeficiency virus.

Twelve rhesus monkeys were vaccinated with SIVmac316 delta nef (lacking nef sequences), and 12 were vaccinated with SIVmac239 delta3 (lacking nef, vpr, and upstream sequences in U3). SIVmac316 and SIVmac239 differ by only eight amino acids in the envelope; these changes render SIVmac316 highly competent for replication in macrophages. Seventeen of the animals developed persistent infections with the vaccine viruses. Seven of the 24 vaccinated animals, however, developed infections that were apparently transient in nature. Six of these seven yielded virus from peripheral blood when tested at weeks 2 and/or 3, three of the seven had transient antibody responses, but none of the seven had persisting antibody responses. The 24 monkeys were challenged in groups of four with 10 rhesus monkey infectious doses of wild-type, pathogenic SIVmac251 at weeks 8, 20, and 79 following receipt of vaccine. None of the seven with apparently transient infections with vaccine virus were protected upon subsequent challenge. Analysis of cell-associated viral loads, CD4+ cell counts, and viral gene sequences present in peripheral blood in the remainder of the monkeys following challenge allowed a number of conclusions. (i) There was a trend toward increased protection with length of time of vaccination. (ii) Solid vaccine protection was achieved by 79 weeks with the highly attenuated SIV239 delta3. (iii) Solid long-term protection was achieved in at least two animals in the absence of complete sterilizing immunity. (iv) Genetic backbone appeared to influence protective capacity; animals vaccinated with SIV239 delta3 were better protected than animals receiving SIV316 delta nef. This better protection correlated with increased levels of the replicating vaccine strain. (v) The titer of virus-neutralizing activity in serum on the day of challenge correlated with protection when measured against a primary stock of SIVmac251 but not when measured against a laboratory-passaged stock. The level of binding antibodies to whole virus by enzyme-linked immunosorbent assay also correlated with protection.

Effect of 3-hydroxyphthaloyl-beta-lactoglobulin on vaginal transmission of simian immunodeficiency virus in rhesus monkeys.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is the major cause of the ongoing AIDS epidemic. Application of chemical barrier methods is expected to contribute to the worldwide control of this epidemic. Bovine beta-lactoglobulin modified by 3-hydroxyphthalic anhydride (3-hydroxyphthalovyl-beta-lactoglobulin [3HP-beta-LG]) was shown to inhibit HIV-1, HIV-2, simian immunodeficiency virus (SIV), herpes simplex virus type 1 and 2, and Chlamydia trachomatis infection in vitro. Here, we show that 3HP-beta-LG not formulated into any vehicle protected three of six rhesus monkeys against vaginal infection by SIV. Incorporation of the compound into an appropriate vehicle is expected to increase the degree of protection. 3HP-beta-LG may be effective as a vaginal inhibitor of HIV-1 infection in humans.

Detection of simian immunodeficiency virus in macaque lymph nodes with a SIVmac envelope probe.

A tritium-labeled DNA envelope gene probe was used to detect Simian Immunodeficiency Virus in formalin fixed lymph nodes from rhesus monkeys experimentally inoculated with SIVmac251. Cells containing SIV RNA produced strong hybridization signal and were present in small numbers in biopsy specimens and in much greater numbers in lymph nodes collected at autopsy. SIV-infected cells were morphologically identified as lymphocytes and macrophages.

Arteriopathy in macaques infected with simian immunodeficiency virus.

BACKGROUND: An arteriopathy characterized by intimal and medial thickening and fibrosis was seen in 19 of 85 rhesus monkeys infected with simian immunodeficiency virus (SIV), a lentivirus with morphologic, genetic, and biologic similarities to HIV-1 and HIV-2. EXPERIMENTAL DESIGN: All cases of simian AIDS in rhesus monkeys at the New England Regional Primate Research Center, resulting from either experimental or naturally acquired SIV infection, were retrospectively examined for evidence of histopathologic changes to the vasculature. Of the 85 SIV-related deaths recorded in the pathology files to date, tissues from 19 animals were chosen for further study because of thickening, disruption, inflammation, or other abnormality to any layer of the vascular wall. The lesion was characterized by special stains, immunoperoxidase procedures, and ultrastructural examination. RESULTS: Affected monkeys of both sexes varied in age from 4 months to 17 years at the time of inoculation and survived from 41 days to 4 years after infection. Pulmonary arteries were affected in all 19 animals, while vessels in other parenchymal organs were involved less frequently. In addition to sometimes marked intimal thickening with luminal occlusion, the internal elastic laminae were fragmented and interrupted. Seven of 19 animals had pulmonary thromboses with varying degrees of organization and recanalization. Immunohistochemical studies, special stains, and ultrastructural analyses revealed the thickened intimae to be composed predominantly of collagen, extracellular matrix, and smooth muscle cells. Ultrastructurally, endothelial cells from both early (no intimal thickening) and advanced lesions were plump, vacuolated, and often disorganized and detached from the subendothelial space. Increased numbers of macrophages (CD68+) were found in the adventitia and occasionally in the thickened intima and media. Rare, fully differentiated macrophages (CD68+, 25F9+) were demonstrated in lumina of affected vessels, some of which expressed p27 SIV gag protein. However, the lesion was not uniformly associated with localization of either viral protein or RNA at the site using immunohistochemistry or in situ hybridization, respectively. A similar arterial lesion has been described in children with AIDS. CONCLUSIONS: The morphologic findings in macaques and their similarity to arteriosclerotic changes induced by experimental endothelial damage in other species collectively suggest that arteriopathy in AIDS may represent a manifestation secondary to primary endothelial injury.

Use of fixed autologous stimulator cells to correctly present human immunodeficiency virus type 1 viral peptides to nonhuman primate lymphocytes in proliferation and cytotoxic T-lymphocyte assays.

Autologous, virus-transformed lymphoblastoid cell lines were established by using peripheral blood lymphocytes from rhesus monkeys that were previously immunized with recombinant human immunodeficiency virus type 1 strain IIIB glycoprotein 160. These autologous cell lines were used to present human immunodeficiency virus type 1 viral antigens in a processed and cell-associated manner to T lymphocytes. This was accomplished by either infecting the cells with recombinant vaccinia viruses or pulsing them with synthetic peptides and then subjecting them to a mild fixation step with glutaraldehyde. Fixed antigen-presenting cells were then used as stimulator cells in vitro to measure cell-mediated immune responses. Both the vaccinia virus-infected and peptide-pulsed autologous cells stimulated antigen-specific cellular proliferative responses. The magnitude of the responses correlated with the immunization histories of the animals and other measures of immunity, such as antibody titers. Autologous vaccinia virus-infected cells were also capable of inducing the in vitro maturation of CD4+ and CD8+ precursor cytotoxic T lymphocytes into antigen-specific mature cytotoxic T lymphocytes. The use of stimulator cells to present viral peptides in a cell-associated manner appeared to be a very sensitive and versatile manner in which to measure cell-mediated immune responses with peripheral blood lymphocytes from nonhuman primates. It is likely that a similar approach will function with peripheral blood lymphocytes from humans.

Protection by live, attenuated simian immunodeficiency virus against heterologous challenge.

We examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV), SIVmac239Delta3, which is missing nef and vpr genes, to protect against challenge by heterologous strains SHIV89.6p and SIVsmE660. SHIV89.6p is a pathogenic, recombinant SIV in which the envelope gene has been replaced by a human immunodeficiency virus type 1 envelope gene; other structural genes of SHIV89.6p are derived from SIVmac239. SIVsmE660 is an uncloned, pathogenic, independent isolate from the same primate lentivirus subgrouping as SIVmac but with natural sequence variation in all structural genes. The challenge with SHIV89.6p was performed by the intravenous route 37 months after the time of vaccination. By the criteria of CD4(+) cell counts and disease, strong protection against the SHIV89.6p challenge was observed in four of four vaccinated monkeys despite the complete mismatch of env sequences. However, SHIV89.6p infection was established in all four previously vaccinated monkeys and three of the four developed fluctuating viral loads between 300 and 10,000 RNA copy equivalents per ml of plasma 30 to 72 weeks postchallenge. When other vaccinated monkeys were challenged with SIVsmE660 at 28 months after the time of vaccination, SIV loads were lower than those observed in unvaccinated controls but the level of protection was less than what was observed against SHIV89.6p in these experiments and considerably less than the level of protection against SIVmac251 observed in previous experiments. These results demonstrate a variable level of vaccine protection by live, attenuated SIVmac239Delta3 against heterologous virus challenge and suggest that even live, attenuated vaccine approaches for AIDS will face significant hurdles in providing protection against the natural variation present in field strains of virus. The results further suggest that factors other than anti-Env immune responses can be principally responsible for the vaccine protection by live, attenuated SIV.

The productive infection of alveolar macrophages by simian immunodeficiency virus.

Alveolar macrophages obtained from healthy rhesus monkeys were infected with SIV in vitro as documented by the appearance of reverse transcriptase activity in the cell-free supernatant, electronmicroscopy, and immunohistochemical methods detecting SIV-related core protein. The results demonstrate permissive infection of alveolar macrophages with SIV in vitro and define a system for studying macrophage-SIV interactions.

Study of long-term cultures of simian immunodeficiency virus (SIVmac 251)-infected peripheral blood lymphocytes.

A culture of rhesus monkey peripheral blood lymphocytes was divided into two parts; one was kept as an uninfected control, and the other was infected with a strain of simian immunodeficiency virus (SIVmac251) originally isolated from a rhesus monkey that died of a malignant lymphoma associated with acquired immune deficiency syndrome. Both cultures were sampled at successive intervals from 1 to 40 days postinfection. Each sample was subjected to in situ hybridization for detection of viral mRNA, immunocytochemical detection of viral core protein (p27), reverse transcriptase assay, electron microscopy, and immunophenotypic characterization of infected cells. These techniques were used to define viral growth kinetics of this novel lentivirus in peripheral blood lymphocytes. The first evidence of SIVmac251 replication was obtained by an in situ hybridization signal for viral mRNA at 2 days postinoculation. This was followed by detection of viral p27 core protein by immunocytochemistry on day 4. Reverse transcriptase activity above control values was not detected until day 8. Budding particles were not found in the infected cultures until 14 days postinfection. Results of in situ hybridization, immunocytochemistry, and reverse transcriptase assay indicated that two bursts of viral replication occurred during the course of this study. The first, at 3 weeks postinfection, was due to infection and subsequent depletion of CD4+ lymphocytes, while the second, 3 weeks later, resulted from a cycle of replication in CD8+ lymphocytes and the remaining CD4+ cells, culminating in the death of all cells on day 39 postinoculation.

Comparative biology of natural and experimental SIVmac infection in macaque monkeys: a review.

Epidemiologic and clinicopathologic data from 11 macaques with naturally acquired SIV infection--10 of which have died--were compared with those from 34 rhesus monkeys that have died of experimental SIVmac infection. Several differences, including gender affected, age at time of death, and the occurrence of certain opportunistic infections, could be explained by the experimental design; others remained unexplained. The most striking difference was the 41% incidence of meningoencephalomyelitis in the experimental group and its absence in naturally SIV-infected animals.

Use of simian immunodeficiency virus for vaccine research.

Rhesus monkeys were immunized with purified, disrupted, noninfectious simian immunodeficiency virus (SIV) in adjuvant induced SIV neutralizing antibodies. Two of six previously vaccinated macaques were protected against infection when challenged with 200-1,000 animal infectious doses of uncloned, pathogenic SIV and both have remained free of signs of virus infection for 19 and 30 months. Prior vaccination appeared to be of benefit in decreasing the virus load and in delaying the onset of AIDS in animals that became infected. Nonetheless, two of four previously vaccinated monkeys that became infected following challenge eventually developed AIDS and died 505 and 538 days after infection. Thus, for a vaccine to be truly effective against AIDS, it may have to protect absolutely against initial infection.

Protection from immunodeficiency virus challenges in rhesus macaques by multicomponent DNA immunization.

Multicomponent DNA vaccines were used to elicit immune responses, which can impact viral challenge in three separate rhesus macaque models. Eight rhesus macaques were immunized with DNA vaccines for HIV env/rev and SIV gag/pol and were challenged intravenously with 10 animal infective doses (AID(50)) of cell-free SHIV IIIB. Three of eight immunized rhesus macaques were protected, exhibiting no detectable virus. Animals protected from nonpathogenic SHIVIIIB challenge were rested for extended periods of time and were rechallenged first with pathogenic SIV(mac239) and subsequently with pathogenic SHIV89.6P viruses. Following the pathogenic challenges, all three vaccinated animals were negative for viral coculture and antigenemia and were negative by PCR. In contrast, the control animals exhibited antigenemia by 2 weeks postchallenge and exhibited greater than 10 logs of virus/10(6) cells in limiting dilution coculture. The control animals exhibited CD4 cell loss and developed SIV-related wasting with high viral burden and subsequently failed to thrive. Vaccinated animals remained virus-negative and were protected from the viral load, CD4 loss, disease, and death. We observed strong Th1-type cellular immune responses in the protected macaques throughout the study, suggesting their important roles in protection. These studies support the finding that multicomponent DNA vaccines can directly impact viral replication and disease in a highly pathogenic challenge system, thus potentially broadening our strategies against HIV.