Microbial translocation in simian immunodeficiency virus (SIV)-infected rhesus monkeys (Macaca mulatta).

BACKGROUND: Chronic immune activation is a hallmark of HIV infection and has been postulated as major factor in the pathogenesis of AIDS. Recent evidence suggests that activation of immune cells is triggered by microbial translocation through the impaired gastrointestinal barrier. METHODS: To determine the association between microbial translocation and disease progression, we have retrospectively analyzed microbial products, viral load and markers of immune activation in a cohort of 37 simian immunodeficiency virus-infected rhesus monkeys, divided in two groups with distinct disease courses. RESULTS: As seen in HIV-infected patients, we found elevated levels of lipopolysaccharide (LPS) in infected animals. However, LPS levels or LPS control mechanisms like endotoxin core antibodies or LPS-binding protein did not differ between groups with different disease progression. In contrast, neopterin, a metabolic product of activated macrophages, was higher in fast progressors than in slow progressors. CONCLUSION: Our data indicate that translocation of microbial products is not the major driving force of immune activation in HIV infection.

The Mhc class II DRB genotype of Macaca fascicularis does not influence infection by simian retrovirus type 2.

Simian retrovirus type 2 (SRV-2) is a natural pathogen of Macaca fascicularis. Although SRV-2 may be endemic in macaque colonies, it is not necessarily detected in all individuals suggesting differential susceptibility to SRV-2; factors contributing to this susceptibility are not fully understood. We have investigated the role of host genetic origin on virus susceptibility. We have shown that high levels of anti-SRV-2 antibodies correlate with failure to establish persistent virus infection, thus we targeted our genetic analysis of virus susceptibility with an investigation of the role of the polymorphic macaque major histocompatibility complex (MHC) class II locus. DRB genotypes, both novel and previously characterised, were identified in individuals and family groups. A discordance with SRV-2 infection status suggests that an Mhc II DRB genotype is not overtly associated with the outcome of viral infection.

Mhc class I haplotypes associated with survival time in simian immunodeficiency virus (SIV)-infected rhesus macaques.

In both human immunodeficiency virus-infected humans and simian immunodeficiency virus (SIV)-infected macaques, genes encoded in the major histocompatibility complex (MHC) class I region are important determinants of disease progression. However, compared to the human human lymphocyte antigen complex, the macaque MHC region encodes many more class I genes. Macaques with the same immunodominant class I genes express additional Mhc genes with the potential to influence the disease course. We therefore assessed the association between of the Mhc class I haplotypes, rather than single gene variants, and survival time in SIV-infected rhesus macaques (Macaca mulatta). DNA sequence analysis and Mhc genotyping of 245 pedigreed monkeys identified 17 Mhc class I haplotypes that constitute 10 major genotypes. Among 81 vaccination-naive, SIV-infected macaques, 71 monkeys carried at least one Mhc class I haplotype encoding only MHC antigens that were incapable of inducing an effective anti-SIV cytotoxic T lymphocytes response. Study of these macaques enabled us to relate individual Mhc class I haplotypes to slow, medium and rapid disease progression. In a post hoc analysis, classification according to disease progression was found to explain at least 48% of the observed variation of survival time.

MhcDRB-sequences from cynomolgus macaques (Macaca fascicularis) of different origin.

Cynomolgus macaques are frequently used in biomedical research. However, in contrast to their closest relative, the rhesus macaque, little is known about their Mhc genes except for the DQB1 locus. In this study, 33 DRB-sequences belonging to 17 allelic lineages were detected in a total of 68 macaques, 58 originating from Mauritius and 10 from China. The majority of the sequences were detected in the few macaques from China, confirming the low degree of genetic variation in macaques from Mauritius. In summary, the DRB region in cynomolgus macaques is polymorphic. The sequences belong in general to the same allelic lineages as in their closest relative, the rhesus macaque. Two exon 2 DNA sequences were identical in both species and may represent a trans-species origin. In addition, protein sequences of members of the DRB*W1 lineage seem to be rather conserved in the three macaque species examined so far. Six DRB-haplotypes were detected in the macaques from Mauritius. While single DRB-alleles or some protein sequences seemed to be conserved among macaque species, we could not detect any evidence for a trans-species conservation of a complete DRB region. Overall, the data indicate that reorganization of the DRB region by recombination is a major force in creating diversity in cynomolgus macaques as it is in rhesus macaques.

Mhc class II DRB sequences of lion-tailed macaques (Macaca silenus).

The lion-tailed macaque (Macaca silenus) is an endangered species. Research into the genetics of this species is important as a basis for coordinated breeding programs of captive populations. Therefore, we sought to analyze the Mhc class II DRB genes of this species because of it is highly polymorphic in genetically heterogeneous populations of most species. Ten individuals from seven families were evaluated. Nine DRB second exon sequences belonging to eight allelic lineages were identified. These lineages are also present in the best-studied macaque species: the rhesus (Macaca mulatta). Although only these relatively few alleles could be isolated, they display variation on the lineage level. This may be a mechanism for increasing their functional diversity.

Sequence-based typing techniques for rhesus macaque MhcMamu-DQB1 allow the identification of more than 35 alleles.

In the recent years, substantial progress has been made in the characterization of the rhesus macaque MHC region, which is more complex than in humans. To cope with the increasing knowledge, improved typing techniques for MHC genes are required. Including the DNA-sequences in this report, 39 rhesus macaque (Mamu)-DQB1 alleles, corresponding to 38 deduced protein sequences, are known. Here, we present a typing technique for Mamu-DQB1 alleles and the DNA-sequences of 9 novel DQB1 alleles. The technique consists of one or two rounds of screening followed by DNA-sequence determination. The first round represents a low-resolution screening sufficiently insensitive to identify novel alleles, which also allows sequencing of both alleles in most heterozygous individuals. The second round consists of a high-resolution screening. This is only necessary for animals in which one PCR-product was generated by the initial screening. The technique was validated by analyzing samples of more than 200 rhesus macaques of different origin, and DNA-sequence determination of more than 230 PCR-products.

Increased reproductive success of MHC class II heterozygous males among free-ranging rhesus macaques.

Gene conversion and balancing selection have been invoked to explain the ubiquitous diversity of the antigen-presenting proteins encoded in the vertebrate major histocompatibility complex (MHC). In the present study, direct evidence for over-dominant selection promoting MHC diversity in primates is provided by the observation that, in a large free-ranging population of rhesus macaques, males heterozygous at MHC class II locus Mamu-DQB1 sired significantly more offspring than homozygotes (the male-specific selection coefficient s equals 0.34). This heterozygote advantage appeared to be independent of the actual male Mamu-DQB1 genotype. No similar effect emerged for a captive group of monkeys of similar genetic background but under veterinary care.

Making the animal model for AIDS research more precise: the impact of major histocompatibility complex (MHC) genes on pathogenesis and disease progression in SIV-infected monkeys.

Experimentally infected rhesus monkeys serve as an indispensable animal model to assess the pathogenesis, to validate therapy approaches and to develop vaccination strategies against viral diseases such as AIDS threatening the human population. Upon infection with simian immunodeficiency virus (SIV), a retrovirus closely related to the human immunodeficiency virus (HIV), macaques develop clinical manifestations similar to those of HIV-infected humans. As in humans, the disease course is variable. Polymorphic genes of the major histocompatibility complex (MHC) are required for the initiation and regulation of a specific immune response and represent a major host factor accounting for the differential outcome of infection. During the last few years, our understanding of the structure and function of the rhesus macaque MHC has increased substantially. Functional studies have led to the identification of specific SIV and HIV peptide epitopes presented by rhesus macaque MHC molecules. The subsequent development of MHC class I tetramers has allowed further insight into the cellular immune response following SIV-infection. Detailed studies demonstrated that viral escape mutants are generated during the acute and chronic phase of infection and explain why control of viral replication ultimately fails. Furthermore, particular MHC haplotypes which influence disease progression have been discovered. Thus, MHC-typing can have a prognostic potential. The further elucidation of the rhesus macaque MHC and the search for other relevant genes will remain an important task for future research and will stimulate all immunologically-related investigations in macaques.

Homozygosity for a conserved Mhc class II DQ-DRB haplotype is associated with rapid disease progression in simian immunodeficiency virus-infected macaques: results from a prospective study.

In human immunodeficiency virus type 1 (HIV-1)-infected individuals, disease progression varies considerably. This is also observed after experimental infection of macaques with simian immunodeficiency virus (SIV). Major histocompatibility complex (MHC) genes may influence disease progression in both species. Homozygosity for Mhc-Mamu (Macaca mulatta)-DQB1*0601 was previously identified to be associated with rapid disease progression in SIV-infected macaques. To validate the association of this genotype with disease progression, a prospective study was carried out. Six unrelated monkeys homozygous for Mamu-DQB1*0601 and DRB1*0309-DRB*W201 and 6 heterozygous monkeys were infected with SIVmac. Five of the homozygous and only 1 of the heterozygous monkeys died rapidly after infection, with manifestations of AIDS. These results were validated by a retrospective survival analysis of 71 SIV-infected monkeys. The identified DQ-DRB genotype is frequent among monkeys of different breeding colonies and allows a fairly reliable selection before infection of monkeys predisposed for rapid disease progression.

Selection of the R17Y substitution in SIVmac239 nef coincided with a dramatic increase in plasma viremia and rapid progression to death.

Three rhesus macaques were infected with an SIVmac239 variant containing substitutions of 73/74PA-->ED and 204D-->R in Nef that disrupted the ability of Nef to downregulate CD4 surface expression. One of these animals, Mm8155, rapidly progressed to AIDS and died 21 weeks postinfection. During the final 5 weeks of infection, the levels of viral RNA and of p27 antigenemia were about 100-fold higher than usually observed in SIVmac239 infection. Postmortem examination revealed giant cell disease of the lymph nodes and the gastrointestinal tract, opportunistic infections, and a severe chronic enteritis. The majority of proviruses in spleen, kidney, and lymph nodes, and almost 100% of the viral RNA sequences, contained mutations of CGA-->TAT in codon 17 of nef, predicting a change of 17R-->Y. The appearance of this substitution, which has recently been shown to confer the phenotype of the acutely pathogenic SIVpbj14, coincided with the dramatic increase in viral load and rapid progression to fatal disease. In comparison, reversions of 204R-->D and changes of 72-74NED-->DKD, which restored the ability of Nef to downregulate CD4, were already selected earlier in infection. Similarly to SIVpbj14, virus reisolated at late time points from Mm8155 replicated efficiently in unstimulated monkey lymphocytes. The Y17 substitution was not detected in 14 additional SIVmac239-infected macaques at the time of AIDS-related death or in the two slowly progressing animals initially infected with the same Nef variant. Although infection of macaques with SIV is commonly used as an animal model for HIV-1 infection in humans, this is only the second example for the emergence of an acutely lethal SIVmac Nef variant.

Mhc-DQ-DRB haplotype analysis in the rhesus macaque: evidence for a number of different haplotypes displaying a low allelic polymorphism.

In the HLA-DRB subregion of man, five major groups of haplotypes, often displaying a remarkable polymorphism, are distinguishable. The polymorphism is thought to be generated by point mutation, microgene conversion and gene rearrangement by recombination. In order to gain insight into the organization of the rhesus macaque major histocompatibility complex (MHC) class II region, DRB genes from monkeys of different origins previously typed for their DQ genes were analyzed. At first DRB haplotypes were deduced from DQ-homozygous monkeys. The stability of these haplotypes was then examined in DQ-heterozygous monkeys by sequence-based typing for the presence of members of the DRB1*03 and DRB1*04 lineage, and for seven single alleles detected on the haplotypes. Six DRB haplotypes linked to the five most frequent and three haplotypes linked to less frequent DQ haplotypes were identified. Six novel DRB alleles were detected. The number of DRB genes per haplotype varied between two and four. The results altogether suggest that in rhesus macaques, in comparison to man, the DQ haplotypes are linked to only a small number of DRB haplotypes, the number and diversity of DRB haplotypes is larger, and the allelic polymorphism of a given haplotype is smaller. The diversity of the DRB haplotypes was partly due to the varying number and identity of genes linked to DRB1*03 and DRB1*04. Furthermore, the number of DRB1 genes themselves varied from zero to two.

Specificity of helper T-cells generated from macaques infected with attenuated simian immunodeficiency virus.

Deletion of the simian immunodeficiency virus (SIV) nef gene leads to an attenuated virus phenotype in vivo. We have previously shown that these viruses induce a potent cellular immune response in macaques. To extend these studies, we established virus-specific short-term T-cell lines from four rhesus macaques infected with a nef deletion mutant of SIV. These T-cell lines proliferated upon restimulation with whole SIV or SIV gp140 antigen in vitro. The proliferating cells were characterized as CD4+ helper T-cells (TH) and their antigen recognition was MHC class II DR-restricted. After antigenic stimulation, they transcribed mRNA for various TH1- and TH2-like cytokines. Using these SIV-specific cell lines, a variety of helper T-cell epitopes in the SIV Env protein were determined with overlapping peptides. TH epitopes were identified throughout the whole SIV Env including both constant and variable regions. Although the recognition of TH epitopes was heterogeneous among different animals, five more broadly reactive T-cell epitopes were identified. As expected, recognition was associated with the MHC class II DRB background of the animals. This is the first report on helper T-cell epitopes in SIV-infected monkeys. Such studies should be of considerable significance for AIDS/ vaccine research.

Paternity assessment in rhesus macaques (Macaca mulatta): multilocus DNA fingerprinting and PCR marker typing.

Establishing kinship relations in primates using modern molecular genetic techniques has enhanced the ability to scrutinize a number of fundamental biological issues. We screened 51 human short tandem repeats (STRs) for cross-species PCR amplification in rhesus macaques (Macaca mulatta) and identified 11 polymorphic loci with heterozygosity rates of at least 0.6. These markers were used for paternity testing in three social groups (M, R, and S) of rhesus macaques from Cayo Santiago, Puerto Rico. Several consecutive birth cohorts were analyzed in which approximately 200 males were tested for paternity against more than 100 mother/ infant pairs. Despite a combined exclusion rate of more than 99.9% in all three groups, some cases could not be solved unequivocally with the STR markers and additional testing of the MHC-associated DQB1 polymorphism. A final decision became possible through multilocus DNA fingerprinting with one or more of the oligonucleotide probes (GATA)4, (CA)8, and (CAC)5. Paternity assessment by multilocus DNA analysis with probe (CAC)5 alone was found to have limitations in rhesus macaques as regards the number of potential sires which might be involved in a given case. Multilocus DNA fingerprinting requires large amounts of DNA, and the ensuing autoradiographic patterns present difficulties in comparisons across gels and even within the same gel across remote lanes. Computer-assisted image analysis was incapable of eliminating this problem. Therefore, a dual approach to DNA typing has been adopted, using STR markers to reduce the number of potential sires to a level where all remaining candidates can be tested by multilocus DNA fingerprinting on a single gel, preferably in lanes adjacent to the mother/infant pair.

DQ-haplotype analysis in rhesus macaques: implications for the evolution of these genes.

The DQA1 and DQB1 alleles of 258 rhesus monkeys (Macaca mulatta) of different origin were typed by PCR-RFLP. Five novel MamuDQA1 and five novel -DQB1 alleles were detected and 15 Mamu-DQA1-DQB1 haplotypes were identified. Haplotype analysis confirmed the conservation of the DQA1*01-DQB1 *06 haplotypes in evolution. The most conspicuous finding was the tight linkage between the Mamu-DQA1 and -DQB1 alleles. Almost in every case the Mamu-DQA1 allele was linked to only one particular Mamu-DQB1 allele. Although there also are constraints in the formation of DQ haplotypes in humans, such tight linkages are not observed. These findings support the hypothesis of some kind of co-evolution between DQA1 and DQB1 alleles and may reflect a stronger force of natural selection in macaques than in humans.

PCR-RFLP-based Mamu-DQB1 typing of rhesus monkeys: characterization of two novel alleles.

Up to now 19 allelic sequences of the rhesus monkey DQB1 locus have been published. Referring to these sequences, we have developed a typing protocol for Mamu-DQB1 alleles which was verified by additional cloning, sequence analysis and segregation studies. The protocol is based on the amplification of the second exon with only one specific primer pair followed by the digestion of the PCR products with up to 10 different restriction endonucleases. The alleles can be identified in homozygous and heterozygous combinations since most amplified second exon sequences give unique hand patterns after digestion with at least one of the selected restriction endonucleases. By the use of this protocol we analyzed DNA-samples from 182 rhesus monkeys. Among these samples two novel Mamu-DQB1 alleles were detected, subsequently cloned and their nucleic sequence determined. Since we typed four complete breeding groups consisting of two generations we were able to identify several DQ haplotypes by segregation analysis using the previously developed typing protocol for DQA1.

Simian immunodeficiency virus (SIV) gp130 oligomers protect rhesus macaques (Macaca mulatta) against the infection with SIVmac32H grown on T-cells or derived ex vivo.

The efficacy of three SIVmac32H gp130 vaccines was compared in rhesus monkeys. Three rhesus monkeys were each immunized over a period of 20 weeks with a total of 600 microgram virion-derived gp130 oligomers (O-gp130) mixed with keyhole limpet hemocyanin and emulsified with incomplete Freund's adjuvant. Three other monkeys were infected with 5 x 10(8) PFU of vaccinia virus wild type (VV-wt) while three additional animals received an equivalent dose of VV expressing the gp130 of SIVmac (VV-gp130). At Week 8, the two VV-wt animals received an additional immunization with 100 microgram O-gp130 each. All VV-infected animals then received booster immunizations at Weeks 12, 16, and 20 with a total of 300 microgram O-gp130 per animal. All animals along with two controls were challenged iv with 50 MID50 of T-cell-grown SIVmac32H at Week 22. Four weeks after the challenge and thereafter, both controls and one animal from either VV group were infected as demonstrated by polymerase chain reaction (PCR), virus isolation, and antibody response. In contrast, all O-gp130 animals and one animal each from the VV-wt and the VV-gp130 group were completely protected as shown by negative PCR and virus reisolation. One animal of the VV-gp130 group was partially protected, since it remained virus isolation negative but became PCR positive. All protected animals did not develop a secondary antibody response. Six months after the first challenge, the five completely protected animals were reimmunized twice 4 weeks apart with a total of 200 microgram O-gp130 per animal. Two weeks later, all animals were challenged with 5 MID50 of the SIVmac32H/spI prepared from the spleen of an immunized, but unprotected SIV-infected rhesus monkey. After the second challenge, all three control animals and one of the vaccinees become productively infected. In contrast, two animals were completely protected, one from the former O-gp130 and one from the former VV-gp130 group. One animal from the former VV-wt group was only DNA-PCR positive and thus partially protected. Therefore, immunization with virion-derived gp130 oligomers of SIVmac32H can confer protection against the infection with T-cell-grown SIVmac32H as well as the ex vivo isolate SIVmac32H/spI.

Cellular immune response of rhesus monkeys infected with a partially attenuated nef deletion mutant of the simian immunodeficiency virus.

To date the vaccines most successful in the simian immunodeficiency virus (SIV) model of AIDS are live attenuated viruses. However, the virus-specific immune response induced after infection of monkeys with attenuated SIV has not been described comprehensively. Therefore, we investigated the cellular immune response of eight rhesus macaques infected with a nef deletion mutant of SIVmac32H (pC8). In contrast to monkeys infected with pathogenic SIV, pC8-infected macaques developed a virus-specific T-cell proliferation. In addition, all animals showed a proliferative T-cell response to recall antigen and mitogens. In six of eight monkeys virus-specific cytotoxic T-cells directed against different SIV polypeptides were detected. In two animals, however, the truncated nef gene reverted to full length 12 weeks after pC8 infection. These two monkeys developed hematological alterations, indicating an immunodeficiency. Simultaneously with the onset of disease the animals lost their T-cell responsiveness against recall antigens. Eight weeks later their T-cell reactivity against mitogens was also abrogated. The results indicate that live attenuated SIV induced a virus-specific cellular immune response in monkeys which might be associated with the previously reported resistance to superinfection with pathogenic SIV. Paradoxically, if the attenuated SIV reverts in vivo to a more virulent virus, the SIV-specific immune response was inefficient to prevent the onset of immunodeficiency in the animals.