Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M).

BACKGROUND: The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H-J5 (rMVA-J5) were assessed. METHODS: Immunization with rMVA constructs (2.5 x 10(7) IU) 32, 20 and 8 weeks pre-challenge was compared with 32 and 20 weeks but with a final boost 8 weeks pre-challenge with 2 x 10(6) fixed-inactivated HSC-F4 cells infected with SIVmac32H. Controls received rMVA vectors expressing an irrelevant transgene or were naïve challenge controls. All received 10 MID(50) SIVmac32H/J5 intravenously. RESULTS: Vaccinates immunized with rMVA-J5 exhibited significant, albeit transient, control of peak primary viraemia despite inconsistent and variable immune responses elicted by vaccination. Humoral and cellular responses to Env were most consistent, with lower responses to Nef, Rev and Tat. Increasing titres of anti-vaccinia neutralizing antibodies reflected the number and dose of rMVA inoculations. CONCLUSIONS: Improved combinations of viral vectors are required to elicit appropriate immune responses to control viral replication.

Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo.

BACKGROUND: A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo. METHODS: SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo. RESULTS: These virus stocks appeared better adapted for replication in M. fascicularis as demonstrated by a greater persistence of recoverable live virus from the periphery and increased pathology in lymphoid tissues 20 weeks post-challenge as detected by immunohistochemistry. Sequence analysis of the envelope gene from these stocks did not identify marked diversification of sequence as a result of this procedure. CONCLUSIONS: These stocks display more robust peripheral persistence and tissue pathology in cynomolgus macaques and should prove valuable analysing recombinant vaccines based upon SIVmacJ5 transgenes.

Specific proliferative T cell responses and antibodies elicited by vaccination with simian immunodeficiency virus Nef do not confer protection against virus challenge.

The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein. This immunization protocol elicited anti-Nef antibodies in all of the vaccinates as well as specific proliferative responses. However, specific cytotoxic T cell responses were not detected before virus challenge. All vaccinates were challenged intravenously with 10 MID(50) of SIVmacJ5 along with four controls. All eight subjects became infected after SIV challenge and there were no group-specific differences in virus load as measured by virus titration and vRNA analysis. The results of this study support indirectly the report from Gallimore and colleagues (Nat Med 1995;1:1667) suggesting that CD8(+) T lymphocyte responses are required for Nef-based vaccines to restrict SIV infection. If Nef-based vaccines are to be beneficial in controlling infection with immunodeficiency viruses, then it will be necessary to develop more effective immunization protocols that elicit potent CD8(+) cell responses reproducibly.

Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359)).

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro.

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV-HXBc2 in human T-cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P < 0.05) to in vitro infection by virulent SIVJ5 and SHIV-HXBc2 than were lymphocytes from naive controls. However, this significant difference in the sensitivity of lymphocytes to virus infection was not observed for more efficiently replicating viruses such as SHIVSF33 and SIVsm3. Virus growth was significantly enhanced (P < 0.01) by depletion of CD8+ T-cells, suggesting a role for these cells in the control of SIV replication, both in vitro and in vivo. We found that levels of the beta-chemokines regulated upon activation, normal T-cell expressed and secreted, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta did not correlate with inhibition of virus replication. Taken together, our findings do not support the hypothesis that retroviral interference is the mechanism by which live attenuated SIVC8 induces protection.

Cloning and sequencing of cynomolgus macaque CCR3, GPR15, and STRL33: potential coreceptors for HIV type 1, HIV type 2, and SIV.

The characterization of several seven-transmembrane G protein-coupled receptors, which function as coreceptors for HIV-1, HIV-2, and/or SIV, has opened up a whole new area of AIDS research. Animal models that have played a central role in the understanding of lentivirus pathogenesis and the design of novel vaccine strategies may also be invaluable in studying the role of these secondary receptors in infection and disease progression. However, since it is known that minor species-specific sequence changes in CCR3 and STRL33 affect their ability to act as coreceptors for HIV-1, HIV-2, and/or SIV, it is important to ascertain whether the relevant receptors function as expected in the animal model of choice. Many studies have been performed on the function of rhesus macaque receptors, but not on the cynomolgus macaque equivalents. Both species are used as animal models for lentivirus pathogenesis, but since there are differences in their susceptibility to viral infection, we felt it was important for information to be available for both rhesus and cynomolgus macaque receptors. The sequence of three cynomolgus macaque receptors, CCR3, GPR15, and STRL33, are presented in this sequence note. These sequences are compared with already published human and rhesus macaque homologs. Functional studies are currently being performed on these three cynomolgus macaque receptors to determine their ability to function as coreceptors for HIV-2, SIV, and/or SHIV isolates.

HIV-1 kills renal tubular epithelial cells in vitro by triggering an apoptotic pathway involving caspase activation and Fas upregulation.

HIV-infected patients suffer several renal syndromes, which can progress rapidly from renal insufficiency to end-stage renal disease. Histologically, HIV-induced nephropathy is characterized by prominent tubulopathy with apoptosis of tubular cells. Clinical and experimental evidence suggests that renal injury may be directly related to virus infection. Although HIV-1 is a polytropic and not solely lymphotropic pathogen, the susceptibility of renal cells to HIV-1 remains to be determined. This paper demonstrates in vitro the permissiveness of proximal tubular epithelial cells (PTEC) to HIV-1 and describes the effects of PTEC infection to explain the pathogenesis of tubular damage in vivo. The results indicate that PTEC express HIV-specific receptor and coreceptors and sustain virus replication. We observed that HIV-1 infection causes the death of tubular cells by triggering an apoptotic pathway involving caspase activation. Fas upregulation but not Fas ligand expression was found in the infected PTEC. However, after HIV-1 infection, tubular cells became susceptible to apoptosis induced through Fas stimulation. Caspase inhibition prevented the death of the infected PTEC in spite of persistent viral replication. These findings may explain the prominent histopathology of HIV-associated nephropathy and demonstrate that the apoptosis of nonlymphoid cells can be directly induced by HIV-1.

VP7 from African horse sickness virus serotype 9 protects mice against a lethal, heterologous serotype challenge.

An established mouse model system was used to evaluate the effectiveness of the major outer core protein VP7 of African horse sickness virus (AHSV) serotype 9 as a subunit vaccine. Balb C mice were immunised with VP7 crystals purified from AHSV infected BHK cells. In groups of mice, each of which was immunised with > or = 1.5 micrograms of the protein in Freund's adjuvant, > or = 80% of mice survived challenge with a virulent strain of a heterologous AHSV serotype (AHSV 7), that killed > or = 80% of the mice in the uninoculated control groups. This level of protection was significantly greater than that observed in mice inoculated with equivalent amounts of either denatured VP7 (50% survival), or GST/VP7 fusion protein (50-70% survival), or which were vaccinated with AHSV 9 (40-50% survival). The VP7 protein folding, or its assembly into crystals, are thought to play some role in the effectiveness of the protective response observed. Titres of circulating antibodies against AHSV VP7 were determined by competitive ELISA but did not appear to correlate with the levels of protection observed. Passive transfer of these antibodies to syngeneic recipients also failed to protect Balb C mice from the AHSV 7 challenge. The observed protection is therefore unlikely to be due to an antibody mediated immune response.

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.

OBJECTIVE: To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep. DESIGN: A clinical and pathological study. PROCEDURE: Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies. RESULTS: All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection. CONCLUSIONS: The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.

African horsesickness virus VP7 sub-unit vaccine protects mice against a lethal, heterologous serotype challenge.

An established mouse model was used to evaluate the effectiveness of the major outer core protein of African horsesickness virus (AHSV), VP7, as a subunit vaccine. Adult female BALB/c mice were immunized with VP7 crystals purified from BHK cells infected with AHSV serotype 9 (AHSV-9), using three inoculations in Freund's adjuvant. Eighty to one hundred per cent of the immunized mice were protected against a heterologous challenge with a known lethal dose of AHSV-7. The protected immunized mice did not develop any clinical signs characteristic of virulent AHSV infection in this model during the study. In contrast, 80-100% mortality was observed in the non-immunized mice that received the same challenge virus. Subsequent studies indicated that a single inoculation of 1.5 micrograms purified AHSV VP7 in Freund's complete adjuvant was sufficient to protect at least 90% of mice from AHSV-7 challenge. If the antigen was presented in the absence of Freund's complete adjuvant, 70% of the mice were still protected by one inoculation of VP7 crystals. Titres of circulating antibody against AHSV VP7, determined by competitive ELISA, did not appear to correlate with protection and passive antibody transfer from immunized BALB/c mice failed to protect syngeneic recipients from AHSV-7 challenge. Therefore, the observed protection is unlikely to be due to an antibody-mediated immune response. The number of viraemic mice and the duration of viraemia post-challenge was significantly reduced in vaccinated mice compared to non-vaccinated controls. However, the levels of viraemia were similar.

Expression of the major core structural protein (VP7) of bluetongue virus, by a recombinant capripox virus, provides partial protection of sheep against a virulent heterotypic bluetongue virus challenge.

A recombinant capripox virus was constructed containing a cDNA copy of genome segment 7 of bluetongue virus (BTV) serotype 1 from South Africa (BTV 1SA), which expressed high levels of the major BTV core protein VP7 in infected lamb testis (LT) cells. Sheep vaccinated with this recombinant virus developed antibodies to VP7 (detected by ELISA) but no neutralizing antibodies to either the homologous or heterologous BTV serotype, prior to challenge (BTV 1 or BTV 3, respectively). Following challenge with a virulent heterotypic strain of BTV (BTV3 SA), all of the animals developed clinical signs of disease, indicating that they were infected and that the challenge virus did replicate. While all of the control animals died, six of the eight animals that were vaccinated with the recombinant capripox virus expressing VP7 recovered fully. This is the first report of a significant level of cross serotype protection against the lethal effects of a challenge with virulent BTV, produced by vaccination with a single BTV core protein, which did not generate a neutralizing antibody response.

Proteolytic cleavage of VP2, an outer capsid protein of African horse sickness virus, by species-specific serum proteases enhances infectivity in Culicoides.

Purified African horse sickness virus (AHSV) was fed, as part of a blood meal, to adult females from a susceptible colony of Culicoides variipennis, established in the insectories at the Institute for Animal Health, Pirbright Laboratory, UK. The meal consisted of heparinized blood obtained from ovine, bovine, equine (horse and donkey) or canine sources spiked with AHSV serotype 9 (AHSV9). The infectivity levels observed for C. variipennis varied significantly, according to the source of the blood sample. Comparison of the protein profiles obtained from AHSV9 incubated with the individual serum of plasma samples indicated that some species-specific serum proteases were able to cleave the outer capsid protein, VP2. The blood samples containing serum proteases that were able to cleave VP2 also showed an increase in infectivity for the insect vector when spiked with purified AHSV.

Application of the polymerase chain reaction to the detection of African horse sickness viruses.

The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA.

Complete nucleotide sequence of RNA segment 3 of bluetongue virus serotype 2 (Ona-A). Phylogenetic analyses reveal the probable origin and relationship with other orbiviruses.

The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them. Three major BTV topotypes (Gould, A.R. (1987) Virus Res. 7, 169-183) were observed which had nucleotide sequences that differed by approximately 20%. At the molecular level, geographic separation had resulted in significant divergence in the BTV genome segment 3 sequences, consistent with the evolution of distinct viral populations. The close phylogenetic relationship between the BTV serotype 2 (Ona-A strain) from Florida and the BTV serotypes 1, 6 and 12 from Jamaica and Honduras, indicated that the presence of BTV serotype 2 in North America was probably due to an exotic incursion from the Caribbean region as previously proposed by Sellers and Maaroof ((1989) Can. J. Vet. Res. 53, 100-102) based on trajectory analysis. Conversely, nucleotide sequence analysis of Caribbean BTV serotype 17 isolates suggested they arose from incursions which originated in the USA, possibly from a BTV population distinct from those circulating in Wyoming.

The use of African horse sickness virus VP7 antigen, synthesised in bacteria, and anti-VP7 monoclonal antibodies in a competitive ELISA.

A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.7% identity at the amino acid level. The cDNA clone was transferred to pGEX-2T (Pharmacia), a bacterial expression vector, such that the reading frame of AHSV9 VP7 was continuous with that of the bacterial glutathione-S-transferase (GST) protein, under the control of the bacterial tac promoter. On induction with IPTG a fusion protein consisting of GST and VP7 was synthesised, which was readily purified on a GST-sepharose column (Pharmacia). The fusion protein reacted equally well in an indirect ELISA using monoclonal antibodies specific for AHSV9 VP7 or polyclonal guinea pig antisera raised against AHSV9 infectious sub-viral particles. This protein was also shown to be a suitable substitute for virus antigen, prepared from infected BHK cell extracts, in a competitive ELISA. Antibodies titres recorded for AHSV9 positive and negative horse sera were similar in the competitive ELISA using either bacterial AHSV VP7 or BHK extracted virus as the source of antigen, in combination with monoclonal or polyclonal antibodies, respectively, as the detectors.

Sequence of genome segment 9 of bluetongue virus (serotype 1, South Africa) and expression analysis demonstrating that different forms of VP6 are derived from initiation of protein synthesis at two distinct sites.

Bluetongue virus (BTV) VP6 is often resolved into two closely migrating bands by SDS-PAGE (VP6 and VP6a). RNA segment 9 of BTV-serotype 1 South Africa (encoding VP6) has been cloned as cDNA, and the complete sequence has been determined. Expression of this clone both in vitro and in tissue culture produced the same polypeptide doublet as seen previously in extracts from BTV-infected cells. Modification of the cDNA, including the removal of the first initiation codon, demonstrated that the two forms of VP6 are derived from initiation of protein synthesis at two distinct sites and not by post-translational modification.

The complete sequence of genome segment 8 of bluetongue virus, serotype 1, which encodes the nonstructural protein, NS2.

Bluetongue virus has a ten-segment double-stranded RNA genome, of which segment 8 encodes a nonstructural protein NS2. This protein is the only bluetongue viral protein to be phosphorylated and also has the ability to bind single-stranded RNA. At present, the function of NS2 is unknown and in order to analyse its characteristics in more detail, it was first necessary to obtain a full-length cDNA clone of the genome segment.

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples.

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complementary to the 3' termini of eight of the ten genome segments were tested. Those representing the 5' termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7. It was confirmed by cloning and sequence analysis, that this PCR-amplified DNA contained both terminal regions of genome segment 7 and therefore represented full length cDNA. Using these segment 7 oligonucleotides it was not only possible to detect routinely as few as 6 molecules of segment 7 dsRNA per sample, but also to detect purified dsRNAs from isolates of other BTV serotypes (1 Australia (AUS), 2, 3, 4, 10, 16 and 20). However, with the exception of Tilligery virus, isolates from other Orbivirus serogroups tested all gave negative results (African horse sickness, epizootic haemorrhagic disease, Palyam, Warrego and Eubenangee). The PCR was also used to analyse red blood cells (RBC) and buffy coat samples from cattle infected with BTV4. Positive results were obtained from samples taken 7 days post-infection (p.i.) (containing 1.6 x 10(3) TCID50 of virus/ml of whole blood) and from the RBC sample only, taken 14 days p.i. (16 TCID50/ml). However, at 28 days p.i. (less than 1.6 TCID50/ml) BTV RNA was not detected using the PCR in either sample.