Sylvie Corbet

env Sequences of Simian Immunodeficiency Viruses from Chimpanzees in Cameroon Are Strongly Related to Those of Human Immunodeficiency Virus Group N from the Same Geographic Area

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) group N from Cameroon is phylogenetically close, in env, to the simian immunodeficiency virus (SIV) cpz-gab from Gabon and SIVcpz-US of unknown geographic origin. We screened 29 wild-born Cameroonian chimpanzees and found that three (Cam3, Cam4, and Cam5) were positive for HIV-1 by Western blotting. Mitochondrial DNA sequence analysis demonstrated that Cam3 and Cam5 belonged to Pan troglodytes troglodytes and that Cam4 belonged to P. t. vellerosus. Genetic analyses of the viruses together with serological data demonstrated that at least one of the two P. t. troglodytes chimpanzees (Cam5) was infected in the wild, and revealed a horizontal transmission between Cam3 and Cam4. These data confirm that P. t. troglodytes is a natural host for HIV-1-related viruses. Furthermore, they show that SIVcpz can be transmitted in captivity, from one chimpanzee subspecies to another. All three SIVcpz-cam viruses clustered with HIV-1 N inenv. The full Cam3 SIVcpz genome sequence showed a very close phylogenetic relationship with SIVcpz-US, a virus identified in aP. t. troglodytes chimpanzee captured nearly 40 years earlier. Like SIVcpz-US, SIVcpz-cam3 was closely related to HIV-1 N inenv, but not in pol, supporting the hypothesis that HIV-1 N results from a recombination event. SIVcpz from chimpanzees born in the wild in Cameroon are thus strongly related inenv to HIV-1 N from Cameroon, demonstrating the geographic coincidence of these human and simian viruses and providing a further strong argument in favor of the origin of HIV-1 being in chimpanzees.

High Levels of Viral Replication during Primary Simian Immunodeficiency Virus SIVagm Infection Are Rapidly and Strongly Controlled in African Green Monkeys

ABSTRACT In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (104 to 105 DNA copies/106 peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 × 106 to 2 × 108 RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 × 105 to 3 × 106 RNA copies/106LN cell [LNC] and 103 to 104 DNA copies/106 LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p.i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (102to 103 DNA copies/106 PBMC and 2 × 103 to 2 × 105 RNA copies/ml of plasma). In LNs, viral loads declined to 5 × 101 to 103 DNA copies and 104 to 3 × 105 RNA copies per 106 LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 × 104 RNA copies/106 LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8+ cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.

Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients

ABSTRACT A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5′-untranslated region (5′-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5′-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5′-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5′-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg.

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.

Routine genotyping of human papillomavirus samples in Denmark

In order to examine a sensitive unbiased consensus PCR with routine sequencing for HPV typing, we analysed Danish male and female patients suspected of having an HPV infection. We used the well‐characterised nested PCR setting with MY09/MY11 and GP5+/GP6+ primers, followed by routine cycle sequencing. Of 1,283 clinical samples from female patients based on suspected HPV infection, we found 379 (29%) negatives and 894 (70%) positives. Samples containing >5000 HPV copies/ml were genotyped by sequencing. Of the 552 HPV genotyped samples from women >15 years of age, 398 were characterised as high‐risk types and the remaining 154 as low‐risk types. The most commonly found high‐risk types were HPV‐16, HPV‐31, HPV‐33, HPV‐18, HPV‐58, and HPV‐52, and the most commonly found low‐risk types were HPV‐6, HPV‐53 and HPV‐11. In addition, we observed that other typing assays could not perform as sensitively or accurately as the nested PCR/cycle sequencing method used in this study. For instance, 87 out of 552 genotyped samples could not have been typed correctly in the Hybrid Capture II assay. Of these 87 samples, 46 (53%) were considered as high‐risk types.

Immune response in rhesus macaques after mixed modality immunisations with DNA, recombinant adenovirus and recombinant gp120 from human immunodeficiency virus type 1†

The establishment of effective regimens for a vaccine against human immunodeficiency virus type 1 (HIV‐1) is urgently needed. In the present study we have produced HIV‐1 gp120 from a vaccine‐relevant primary R5 isolate in recombinant vaccinia (rVV)‐infected Vero cells. We have investigated the effect of boosting with this protein in mixed modality immunisations of rhesus macaques following different immunisation. As reported earlier, animals were primed with codon‐optimised HIV‐1BX08env DNA delivered as plasmid or as replication‐deficient recombinant human adenovirus type 5 (rAd5), which both induced specific antibody and cellular immune responses (1). Boosting with rAd5 temporarily had increased the anti‐gp120 antibody titres approximately 1 log (rAd5+rAd5) or 3 log (DNA+rAd5) (1). However, secondary rAd5 boosting showed less effect due to the induced vector‐specific immunity. To further boost the antibody response, the rgp120BX08 was injected with Quadri A saponin adjuvant. The protein boosting resulted in a 1–2 log antibody increase and also boosting of the cell‐mediated immune response. Neutralising antibodies to the heterologous HIV‐1MN were detected; however, neutralising antibodies to the primary HIV‐1Bx08 isolate were seen only transiently after rAd5 but not the rgp120 immunisation. It is concluded that the rgp120Bx08 reagent from rVV‐infected Vero cells is functional and immunogenic in macaques, inducing both antibody and cellular immunity. The rgp120Bx08 is a relevant model antigen that may be used to boost antibody and cellular immunity in mixed modality vaccine regimens against HIV‐1 in higher animals.

Mutations in CCR5-coding sequences are not associated with SIV carrier status in African nonhuman primates.

African monkeys can be naturally infected with SIV but do not progress to AIDS. Since mutations in the human CCR5 gene have been shown to influence susceptibility to HIV infection and disease progression, we have now investigated whether mutations in CCR5-coding sequences in African nonhuman primates can explain species-specific differences in susceptibility to lentiviral infection. The animals studied comprise chronically infected monkeys corresponding to four natural hosts of SIV (Cercopithecus aethiops, Cercopithecus pygerythrus, Cercopithecus sabaeus, and Cercopithecus tantalus), noninfected animals from three species that are known to be susceptible to SIV infection (Cercopithecus patas, Cercopithecus Ihoesti, and Pan troglodytes), and monkeys of six species that do not carry SIV in the wild (Cercocebus galeritus, Cercocebus aterrimus, Cercopithecus ascanius, Cercopithecus nictitans, Cercopithecus neglectus, and Cercopithecus cephus). We observed a high degree of genetic divergence among the species. The rate of accumulation of amino acid mutations was, however, not higher in SIV carriers than in other nonhuman primates. No homozygous premature stop codons, deletions, or frameshift mutations were detected. In at least two animals, one infected AGM (Cercopithecus tantalus) and one noninfected monkey (Cercocebus aterrimus), the CCR5 alleles identified encode functional proteins, as they were identical in terms of amino acid sequence to that of functional CCR5 reported in the literature. We found no other consistent differences in the genetic variability of CCR5-coding sequences between the nonhuman primates that are carriers of SIV and those that are not.

Sequence conservation of subdominant HLA‐A2‐binding CTL epitopes in HIV‐1 clinical isolates and CD8+ T‐lymphocyte cross‐recognition may explain the immune reaction in infected individuals

Cytotoxic T‐lymphocytes (CTL) are critical for immune control of infection with human immunodeficiency virus type‐1 (HIV‐1) and searches for relevant CTL epitopes for immune therapy are ongoing. Recently, we identified 28 HLA‐A2‐binding HIV‐1 CTL epitopes (1). In this follow‐up study we fully genome sequenced HIV‐1 from 11 HLA‐A2+ patients to examine the sequence variation of these natural epitopes and compared them with the patients CD8+ T‐cell recall response. Often the epitope was conserved but only a few patients showed a CD8+ T‐cell recall response. This infrequent targeting may be explained by immune subdominance. CD8+ T‐cell recall response to a natural epitope could be measured despite sequence differences in the patients virus. T‐cell cross‐reaction between such variants could be demonstrated in HLA‐A2 transgenic mice. Nine infrequently targeted but conserved or cross‐reacting epitopes were identified in seven HIV‐1 proteins. More immunogenic anchor amino acid optimized immunogens were designed that induced T‐cell cross‐reaction with these natural epitopes. It is concluded that most of the new CTL epitopes are conserved but subdominant during the infection. It is suggested that T‐cell promiscuity may explain the observed CD8+ T‐cell reaction to epitope variants and it may be possible to use the selected immune optimized epitope peptides for therapeutic vaccination.

Genetic subspecies diversity of the chimpanzee CD4 virus-receptor gene

Chimpanzees are naturally and asymptomatically infected by simian immunodeficiency virus (SIV). Pathogenic properties of SIV/HIV vary and differences in susceptibility and pathogenicity of SIV/HIV depend in part on host-specific factors such as virus-receptor/co-receptor interactions. Since CD4 plays a primary role in virus binding and since SIVcpz have been found only in two African chimpanzee subspecies, we characterized the genetic diversity of CD4 receptors in all four recognized subspecies of chimpanzees. We found noticeable variation in the first variable region V1 of CD4 and in intron six among the subspecies of chimpanzees. We found the CD4 receptor to be conserved in individuals belonging to the P. t. verus subspecies and divergent from the other three subspecies, which harbored highly variable CD4 receptors. The CD4 receptor of chimpanzees differed from that of humans. We question whether the observed diversity can explain the species-specific differences in susceptibility to and pathogenicity of SIV/HIV.