Antibody-mediated protection against MERS-CoV in the murine model.

Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.

Mhc haplotype M3 is associated with early control of SHIVsbg infection in Mauritian cynomolgus macaques.

The restricted major histocompatibilty complex of Mauritian cynomolgus macaques confers exceptional potential on this species in human immunodeficiency virus (HIV) vaccine development. However, knowledge of the effects of Mhc genetics on commonly used simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) stocks is incomplete. We determined the effect of Mhc haplotypes on SHIVsbg replication kinetics in a cohort of 25 naïve cynomolgus macaques. Haplotype M3 was associated with a 1.58log(10) reduction in viraemia at day 28 post infection (p.i.). Haplotype M6 was associated with elevated SHIVsbg viraemia at days 28 and 56. No significant effect of Mhc class II haplotypes on viral replication was observed. These data emphasise the importance of genetic characterisation of experimental macaques and advance our understanding of host genetic effects in SIV/SHIV models of HIV infection.

Spectral signatures of the surface reconstructions of Au(110)/electrolyte interfaces.

It is demonstrated that the (1 × 1) structure and the (1 × 2) and (1 × 3) surface reconstructions that occur at Au(110)/electrolyte interfaces have unique optical fingerprints. The optical fingerprints are potential, pH and anion dependent and have potential for use in monitoring dynamic changes at this interface. We also observe a specific reflection anisotropy spectroscopy signature that may arise from anions adsorbed on the (1 × 1) structure of Au(110).

Low rates of transmission of SRV-2 and STLV-I to juveniles in a population of Macaca fascicularis facilitate establishment of specific retrovirus-free colonies.

BACKGROUND: Prevalence of simian retrovirus-2 (SRV-2) and simian T lymphotropic virus type I (STLV-I), was unknown in 337 captive cynomolgus macaques. METHODS AND RESULTS: Molecular assays identified 29% of animals as SRV-2 mono-infected, 4% of animals as STLV-I mono-infected and 9% of animals as dual-infected. Of 108 juvenile animals, 83% were SRV-2-negative and no juvenile animal was STLV-I-positive. A subsequent study of juvenile macaques over a period of 2.5 years detected no STLV-I and 10 SRV-2 infections, six of which occurred between testing and day of colony formation. The study also highlighted that an anti-SRV-2 serological response does not presuppose infection. Tissue reservoirs of latent SRV-2 were not identified in suspected SRV-2 infections. CONCLUSIONS: Low transmissibility of the viruses present in the parental cohort and improved knowledge of the host response to SRV-2 has facilitated the creation of specific-retrovirus-free colonies of cynomolgus macaques.

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.

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.

Orientation of ordered structures of cytosine and cytidine -monophosphate adsorbed at Au(110)/liquid interfaces.

It is demonstrated using reflection anisotropy spectroscopy that the adsorption of cytosine and cytidine -monophosphate at the Au(110) 1 x 2/electrolyte interface gives rise to ordered structures in which the base is oriented vertical to the surface and parallel to the [110] axis of the Au(110) plane.

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.

Simian immunodeficiency virus Nef gene regulates the production of 2-LTR circles in vivo.

The replication dynamics of simian immunodeficiency virus (SIVmac32H-C8), attenuated through discrete genetic disruption of the nef gene, were compared with the wild-type parental clone (SIVmac32H-J5) using quantitative molecular methods. The primary viraemia of both infections were similar during the first week, but peaked on Day 10 at higher levels for wild-type virus. Viral RNA levels differed most markedly at Day 14. The frequency and levels of viral DNA species, detectable as gag provirus or circular 2-LTR episomes, differed depending on the virus and the lymphoid compartment sampled. 2-LTR circles persisted for prolonged periods in the peripheral blood but were never detected in any SIVmac32H C8-infected tissue, even if positive by gag PCR. Paradoxically, the converse was observed following wild-type infection. 2-LTR circles disappeared from the peripheral blood by Day 42 postinfection but persisted in lymphoid tissues. These findings are discussed in terms of nef and the role and stability of 2-LTR circle forms in vivo.

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.

Comparison of early plasma RNA loads in different macaque species and the impact of different routes of exposure on SIV/SHIV infection.

Various simian immunodeficiency virus (SIV)sm/mac and simian/human immunodeficiency virus (SHIV) strains are used in different macaque species to study AIDS pathogenesis, as well as to evaluate candidate vaccine and anti-retroviral drugs efficacy. In this study we investigated the effect of route of infection, species of macaques and nature of virus stock on early plasma viral RNA load. We monitored the plasma RNA concentrations of 63 rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) infected with well-characterised virus stocks administered either by oral, rectal, vaginal or intravenous (i.v.) routes. In SIV(mac)-infected macaques, no significant difference in plasma RNA loads was observed between the rectal, oral and i.v. routes of infection. Cynomolgus macaques developed lower steady state SIV plasma RNA concentrations compared with rhesus macaques and no significant difference was observed between rectal and i.v. routes of infection. In SHIV(89.6p)-infected macaques, no difference between species or between route of infection was observed with this particular chimeric virus.

'Chemical condoms' for the prevention of HIV infection: evaluation of novel agents against SHIV(89.6PD) in vitro and in vivo.

BACKGROUND: Vaginal agents which are antiviral and/or inhibit the entry of HIV into the cell could prevent heterosexual transmission of HIV, and protect women who cannot negotiate condom use. METHODS: Four agents have been investigated for activity in vitro and in vivo against SHIV(89.6PD): two anionic polymers, dextrin-2-sulphate (D2S) and PRO 2000 (P2K), and two virucidal agents; a non-ionic detergent, nonoxynol-9 (N9) and a cyclic peptide ionophore, gramicidin-D (GD). All four agents were investigated in rhesus macaques, using an intra-vaginal challenge of two inoculations of 1 x 104 50% tissue culture infectious doses (TCID)50 of SHIV(89.6PD). RESULTS: D2S, P2K, GD and N9 all inhibited SHIV(89.6PD) in vitro. In vivo, three out of four control macaques were infected as judged by viral culture, seroconversion, DNA and RNA PCR; infection was confirmed in four out of eight macaques pre-treated with P2K, two out of four pre-treated with D2S, one out of four pre-treated with N9, two out of four pre-treated with GD and four out of four pre-treated with D2S + GD, a combination additive in vitro. INTERPRETATION: D2S and PRO-2000, novel inhibitors of HIV entry, showed evidence of protection in vivo, comparable to that seen with the virucide, N9. These data, together with the results of phase I and phase II studies in healthy women which have shown minimal toxicity, support plans for a phase III efficacy trial of chemically simple inhibitors of HIV entry with low toxicity, for the prevention of HIV infection in women.

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.

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes.

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.

Construction of infectious SIV/HIV-2 chimeras.

OBJECTIVE: To construct SIV/HIV-2 chimeras (SHIV) that replicate in vivo. These would be valuable tools to elucidate the mechanism by which HIV-2 can bypass protection conferred by live attenuated SIV vaccines. METHOD: Novel SHIV were constructed to express either the vpx, vpr, tat, rev and env genes (SHIV-2isy env) or the gag and pol genes (SHIV-2isy gag/pol) of the infectious molecular clone HIV-2isy in an SIVmac backbone. The replication of SHIV-2isy env and SHIV-2isy gag/pol were evaluated on selected cell lines and peripheral blood mononuclear cells (PBMC) in vitro. In addition, their infectivity was assessed in vivo. RESULT: Virus stocks of SHIV-2isy env and SHIV-2isy gag/pol were prepared in vitro. For SHIV-2isy gag/pol both the 5' and 3' boundaries of the chimeric construct were critical for infectivity in vitro. The growth of each chimera on T cell lines in vitro mirrors that of the parental viruses donating the envelope gene. On PBMCs SHIV-2isy env replicated well on human and simian PBMC whereas SHIV-2isy gag/pol replicated to detectable levels on human PBMC only. In vivo, SHIV-2isy env virus was isolated from one of two cynomolgus macaques challenged intravenously, SHIV-2isy gag/pol was isolated from one of two cynomolgus macaques and both rhesus macaques challenged intravenously. CONCLUSION: This is the first report of SIV/HIV-2 chimeras that are infectious in macaques. Moreover, this is the first report of an infectious chimera in which both SIV gag and pol have been replaced with the equivalent regions of an HIV isolate.

The appearance of escape variants in vivo does not account for the failure of recombinant envelope vaccines to protect against simian immunodeficiency virus.

The presence or evolution of immune escape variants has been proposed to account for the failure of recombinant envelope vaccines to protect macaques against challenge with simian immunodeficiency virus (SIVmac). To address this issue, two groups of three cynomolgus macaques were immunized with recombinant SIV Env vaccines using two different vaccine schedules. One group of macaques received four injections of recombinant SIV gp120 in SAF-1 containing threonyl muramyl dipeptide as adjuvant. A second group were primed twice with recombinant vaccinia virus expressing SIV gp160 and then boosted twice with recombinant SIV gp120. Both vaccine schedules elicited neutralizing antibodies to Env. However, on the day of challenge, titres of anti-Env antibodies measured by ELISA were higher in macaques primed with recombinant vaccinia virus. Following intravenous challenge with 10 monkey infectious doses of the SIVmac J5M challenge stock, five of the six immunized macaques and all four naive controls became infected. The virus burdens in PBMC of macaques that were primed with recombinant vaccinia virus were lower than those of naive controls, as determined by virus titration and quantitative DNA PCR. Sequence analysis was performed on SIV env amplified from the blood of immunized and naive infected macaques. No variation of SIV env sequence was observed, even in macaques with a reduced virus load, suggesting that the appearance of immune escape variants does not account for the incomplete protection observed. In addition, this study indicates that the measurement of serum neutralizing antibodies may not provide a useful correlate for protection elicited by recombinant envelope vaccines.

Monoclonal antibodies recognize at least five epitopes on the SIV Nef protein and identify an in vitro-induced mutation.

Eleven monoclonal antibodies (MAbs) to SIV Nef were produced and characterized. Five antibody-binding sites on SIV Nef were identified on the basis of the reactivity of the antibodies with recombinant proteins. Two of the five epitopes were defined using overlapping peptides. A further three epitopes could not be defined with peptides but all antibodies reacted in Western blot, suggesting that the epitopes were at least partially conformation dependent. Antibodies in two of the five epitope groups were further differentiated by competition analysis. The panel of MAbs described is able to distinguish between a number of recombinant Nef proteins currently under investigation in vivo in macaques. Two of the MAbs described are able to distinguish between the Nef protein from pathogenic (J5) and attenuated (C8) strains of SIV, thus providing useful tools for studying the relevance of the Nef protein in the pathogenesis of SIV infection. In FACScan analysis two of the MAbs, KK70 and KK75, were used to identify an in vitro-induced mutation in J5 Nef grown in C8166 cells. Sequence analysis of the phenotypic variants identified a mutation of the tryptophan (TGG) at amino acid 214 to a stop codon (TGA), thus truncating the Nef protein. The functional significance of this observation remains unclear but highlights the need to interpret data with caution if virus has been cultured in vitro even for a short period of time.