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Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2beta2-2beta3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region. Overall, four out of six rabbits vaccinated with the MAP peptide in Freund's adjuvant developed high-titer, high-avidity antibody responses which cross-reacted with the immobilized peptide sequence comprising aa 305 to 319 and with PA, as determined by an enzyme-linked immunosorbent assay, and which displayed potent and durable neutralization of lethal toxin (LeTx) in vitro, with peak titers which were 452%, 100%, 67%, and 41% of the peak neutralization titers observed in positive-control rabbits immunized with PA. Importantly, analysis of sera from multiple cohorts of rabbits with high-titer immunity to PA demonstrated a virtual absence of this potent antibody specificity, and work by others suggests that this specificity may be present at only low levels in primate PA antiserum. These results highlight the potential importance of this immunologically cryptic neutralizing epitope from PA as a target for alternative and adjunctive vaccines for anthrax.

Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (approximately 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.

Gene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.

Passively transferred neutralizing antibodies can block lentivirus infection, but their role in postexposure prophylaxis is poorly understood. In this nonhuman-primate study, the effects of short-term antibody therapy on 5-year disease progression, virus load, and host immunity were explored. We reported previously that postinfection passive treatment with polyclonal immune globulin with high neutralizing titers against SIVsmE660 (SIVIG) significantly improved the 67-week health of SIVsmE660-infected Macaca mulatta macaques. Four of six treated macaques maintained low or undetectable levels of virus in plasma, compared with one of ten controls, while two rapid progressors controlled viremia only as long as the SIVIG was present. SIVIG treatment delayed the de novo production of envelope (Env)-specific antibodies by 8 weeks (13). We show here that differences in disease progression were also significant at 5 years postinfection, excluding rapid progressors (P = 0.05). Macaques that maintained

Evolution of the domain encoding the V1/V2 variable region of the simian immunodeficiency virus sm (SIVsm) envelope (env) gene was analyzed in relation to route of virus challenge, virus load, and neutralizing antibody (NAb) titers during primary infection of rhesus macaques with the pathogenic SIVsmE660 isolate. In this model system animals are initially infected with multiple viruses as evidenced by the presence of multiple V1/V2 genotypic variants that could be resolved by using a heteroduplex tracking assay (HTA). Overlapping subsets of the multiple variants were established in each animal. There was no selection for the establishment of specific variants in comparing intravenous- and intrarectal-challenged macaques at week 2 postinfection, suggesting that no genotypic selection occurred at the mucosal surface. There was an initial period of significant stability of the V1/V2 variants. Macaques challenged intravenously displayed subsequent V1/V2 diversification significantly earlier than macaques challenged intrarectally and well past the initial resolution of viremia. The time when SIVsmE660-specific NAbs reached a threshold titer of 100 was significantly correlated with the timing of V1/V2 diversification, even though antibodies to the Env protein could be detected much earlier. The time when NAbs reached a titer of 400 was significantly correlated with virus load late in infection. These results show that the route of infection affects the timing of V1/V2 diversification and that this diversification is correlated with the maturation of a specific NAb response. However, prior immunization capable of priming an anamnestic Env antibody response did not accelerate V1/V2 diversification. This result suggests that diversification of the SIV env V1/V2 region is the result of a type-specific antibody response.

Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.

Although several human immunodeficiency virus (HIV) vaccine approaches have elicited meaningful antigen-specific T-cell responses in animal models, no single vaccine candidate has engendered antibodies that broadly neutralize primary isolates of HIV type 1 (HIV-1). Thus, there remains a significant gap in the design of HIV vaccines. To address this issue, we exploited the existence of rare human monoclonal antibodies that have been isolated from HIV-infected individuals. Such antibodies neutralize a wide array of HIV-1 field isolates and have been shown to be effective in vivo. However, practical considerations preclude the use of antibody preparations as a prophylactic passive immunization strategy in large populations. Our concept calls for an antibody gene of choice to be transferred to muscle where the antibody molecule is synthesized and distributed to the circulatory system. In these experiments, we used a recombinant adeno-associated virus (rAAV) vector to deliver the gene for the human antibody IgG1b12 to mouse muscle. Significant levels of HIV-neutralizing activity were found in the sera of mice for over 6 months after a single intramuscular administration of the rAAV vector. This approach allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein.

Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.

Cytotoxic T lymphocyte (CTL) responses against the simian immunodeficiency virus (SIV) envelope and Gag proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC-->M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8(+) T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.

A reliable method for the quantitation of plasma viremia in nonhuman primates infected with simian immunodeficiency virus (SIV) and related viruses is described. This method is based on an established quantitative-competitive PCR format and includes a truncated control for internal assay calibration. Optimization of assay conditions has significantly improved amplification specificity, and interassay variability is comparable to that of commercially available assays for human immunodeficiency virus (HIV) quantitation. This procedure was used to monitor viral loads in a group of Macaca mulatta animals that were infected with SIVsmE660 for over 2 years. Highly diverse profiles of plasma viremia were observed among animals, and high viral loads were associated with more rapid disease progression. Spearman rank correlation analyses were done for survival versus three parameters of viral load: plasma viremia, p27 core antigen, and frequency of infected peripheral blood mononuclear cells. Plasma viremia had the strongest overall correlation and was significantly (P < 0.05 to P < 0.01) associated with survival at 10 of the 13 time points examined. Plasma viremia did not correlate with survival during the primary viremia phase; however, the strength of this correlation increased with time postinfection and, remarkably, viremia levels as early as week 6 postinfection were highly predictive (P < 0.01) of relative survival. These findings are consistent with the available clinical data concerning viral load correlates early in HIV infection, and they provide further support for the view that disease outcome in lentiviral infection may be largely determined by events that occur shortly after infection.

Previous studies suggested that simian immunodeficiency viruses isolated from African green monkeys (SIVagm) are relatively nonpathogenic. The report describes the isolation and biologic and molecular characterization of a pathogenic SIVagm strain derived from a naturally infected African green monkey. This virus induced an AIDS-like syndrome characterized by early viremia, frequent thrombocytopenia, severe lymphoid depletion, opportunistic infections, meningoencephalitis, and death of five of eight macaques within 1 year after infection. An infectious clone derived from this isolate reproduced the immunodeficiency disease in pig-tailed (PT) macaques, providing definitive proof of the etiology of this syndrome. Although the virus was highly pathogenic in PT macaques, no disease was observed in experimentally infected rhesus macaques and African green monkeys despite reproducible infection of the last two species. Whereas infection of PT macaques was associated with a high viral load in plasma, peripheral blood mononuclear cells, and tissues, low-level viremia and infrequent expression in lymph nodes of rhesus macaques and African green monkeys suggest that differences in pathogenicity are associated with the extent of in vivo replication. The availability of a pathogenic molecular clone will provide a useful model for the study of viral and host factors that influence pathogenicity.

Simian immunodeficiency virus (SIV) exists within tissues of infected macaques as a mixture of diverse genotypes. The goal of this study was to investigate the biologic significance of this variation in terms of cellular tropism and pathogenicity. PCR was used to amplify and clone 3'-half genomes from the spleen of an immunodeficiency SIV-infected pig-tailed macaque (Macaca nemestrina). Eight infectious clones were generated by ligation of respective 3' clones into a related SIVsm 5' clone, and virus stocks were generated by transient transfection. Four of these viruses were infectious for macaque peripheral blood mononuclear cells (PBMC) or monocyte-derived macrophages (MDM). Three viruses with distinct tropism for macaque PBMC or MDM were tested for in vivo infectivity and pathogenicity. The ability of these three viruses to infect PBMC and macrophages correlated with differences in infectivity and pathogenicity. Thus, a virus that was infectious for both PBMC and MDM was highly infectious for macaques and induced AIDS in half of the inoculated animals. In contrast, virus that was less infectious for PBMC and not infectious for MDM induced only transient viremia. Finally, a virus that was not infectious for either primary cell type did not infect macaques. Chimeric clones exchanging portions of the envelope gene of the 62A and smH4 molecular clones and a series of point mutants were used to map the determinant of tropism to a 60-amino-acid region of gp120 encompassing the V3 analog of SIV. Naturally occurring mutations within this region were critical for determining tropism and, as a result, pathogenicity of these SIVsm clones.

Simian immunodeficiency virus (SIV) induces an immunodeficiency syndrome similar to human AIDS. Although the disease course of SIV-induced immunodeficiency is generally measured in months to years, a disease syndrome that results in death in 5 to 14 days has been described in pig-tailed macaques infected with the SIVsmmPBj (PBj) strain. The purpose of this study was to derive an acutely lethal PBj molecular clone in order to study viral genes involved in pathogenesis. Six infectious molecular clones were generated; acutely fatal disease was induced by experimental inoculation of pig-tailed macaques with virus stocks derived from either of two clones, PBj6.6 or PBj14.6. Molecular chimeras were constructed by exchange of regions of the genome of PBj6.6 and a nonlethal, related clone, SIVsmH4. Only a chimera expressing the PBj genome under the control of a SIVsmH4 long terminal repeat induced death soon after inoculation. These studies suggest that multiple viral genes of PBj are critical for development of acute disease. More specifically, the env gene but not the long terminal repeat PBj was required for acute disease induction; however env must act in concert with another gene(s) of the PBj genome.

Asymptomatic infection with simian immunodeficiency virus (SIV) has been demonstrated in African Sykes' monkeys (Cercopithecus mitis albogularis), and virus isolation confirmed infection with a novel SIV from Sykes' monkeys (SIVsyk). Macaques inoculated with SIVsyk became persistently infected but remained clinically healthy. We utilized polymerase chain reaction amplification to generate a full-length, infectious molecular clone of SIVsyk. The genome organization of SIVsyk is similar to that of the other primate lentiviruses, consisting of gag, pol, vif, vpr, tat, rev, env, and nef. A unique feature is the absence of the highly conserved NF-kappa B binding site in the long terminal repeat. SIVsyk is genetically equidistant from other primate lentiviruses. Thus, SIVsyk represents a new group that is distinct from the four previously recognized primate lentivirus groups: human immunodeficiency virus type 1 (HIV-1), SIV from sooty mangabeys (SIVsmm) and HIV-2, SIV from African green monkeys (SIVagm), and SIV from mandrills (SIVmnd). The genetic differences between SIVsyk and SIVagm, isolates derived from monkeys of the same genus, underscore the potential for other distinct SIVs which have yet to be isolated and characterized.

The prevalence, natural history, and genetic characteristics of simian immunodeficiency virus (SIV) infections in most feral African monkey species are presently unknown, yet this information is essential to elucidate their origin and relationship to other simian and human immunodeficiency viruses. In this study, a combination of classical and molecular approaches were used to identify and characterize SIV isolates from West African green monkeys (Cercopithecus sabaeus) (SIVagm isolates). Four SIVagm viruses from wild-caught West African green monkeys were isolated and analyzed biologically and molecularly. Amplification, cloning, and sequencing of a 279-bp polymerase fragment directly from uncultured peripheral blood mononuclear cells was facilitated by the use of nested polymerase chain reaction. The results indicated that West African green monkeys are naturally infected with SIVs which are closely related to East African SIVagm isolates. However, structural, antigenic, and genetic differences were observed which strongly suggest that the West African green monkey viruses comprise a phylogenetically distinct subgroup of SIVagm. These findings support our previous hypothesis that SIVagm viruses may have evolved and diverged coincident with the evolution and divergence of their African green monkey host. In addition, this study describes a polymerase chain reaction-based approach that allows the identification and molecular analysis of divergent SIV strains directly from primary monkey tissue. This approach, which does not depend on virus isolation methods, should facilitate future studies aimed at elucidating the origins and natural history of SIVs in feral African green monkey populations.

African green monkeys are asymptomatic carriers of simian immunodeficiency viruses (SIV), commonly called SIVagm. As many as 50% of African green monkeys in the wild may be SIV seropositive. This high seroprevalence rate and the potential for genetic variation of lentiviruses suggested to us that African green monkeys may harbor widely differing genotypes of SIVagm. To investigate this hypothesis, we determined the entire nucleotide sequence of an infectious proviral molecular clone of SIVagm (155-4) and partial sequences (long terminal repeat and Gag) of three other distinct SIVagm isolates (90, gri-1, and ver-1). Comparisons among the SIVagm isolates revealed extreme diversity at the nucleotide and amino acid levels. Long terminal repeat nucleotide sequences varied up to 35% and Gag protein sequences varied up to 30%. The variability among SIVagm isolates exceeded the variability among any other group of primate lentiviruses. Our data suggest that SIVagm has been in the African green monkey population for a long time and may be the oldest primate lentivirus group in existence.

Plasma from two rhesus macaques (Macaca mulatta) experimentally infected with the simian immunodeficiency virus (SIV; isolate SIVmac251) enhanced SIVmac infection of a human CD4+ lymphoblastoid cell line, MT-2. Prechallenge plasma samples from these animals and serum from SIV-negative macaques did not enhance infection. Compared with controls, infection enhancement was characterized by the rapid appearance of syncytium formation (3 to 4 days sooner), reverse transcriptase release (10-fold increase), and cytopathic effect (60% cell killing). Enhancement of activity was dependent on the presence of diluted, fresh SIV-negative macaque serum as a source of complement. A requirement for complement was shown by the absence of enhancement in heat-inactivated serum and by dose-dependent inhibition of enhancement in the presence of polyclonal antibody to monkey complement component C3. Monoclonal antibody to CD4 (OKT4a) blocked enhancement completely, while monoclonal antibody to the human complement component C3d receptor CR2 (OKB7) reduced enhancement by greater than 50%, indicating a requirement for CD4 and CR2 in mediating this phenomenon. SIV infection-enhancing activity appeared in macaques soon after experimental inoculation (28 days). The titer increased over time and peaked just prior to the death of both macaques from opportunistic infections and lymphoma. In vitro SIV infection enhancement is nearly identical to the in vitro complement-mediated, antibody-dependent enhancing (C'-ADE) activity observed in human immunodeficiency virus-positive human sera (Robinson et al., Lancet i:790-794, 1988; Robinson et al., J. Acq. Immun. Def. Synd. 2:33-42, 1989). These observations validate the macaque-SIV model for studies of C'-ADE.

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus.