T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates.

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.

Canine hepacivirus is not associated with chronic liver disease in dogs.

Canine hepacivirus (CHV) has recently been identified in liver and respiratory tract samples from dogs, and comparative phylogenetic analysis has confirmed it to be the closest genetic relative of hepatitis C virus (HCV) described to date. CHV offers great potential as a model system for HCV, but only if the underlying processes of infection and pathogenesis are similar for both viruses. However, it is not yet clear if CHV is hepatotrophic. Canine chronic hepatitis (CH) is a common and usually idiopathic disease that shares similar histological features to that of HCV infection of humans. To date, no study has attempted to determine whether CHV is involved in the aetiology of liver disease in dogs. We employed two nested PCR assays, using primers targeting regions of the helicase domain of CHV NS3, to identify viral nucleic acids in liver samples from 100 dogs with CH of unknown cause in the UK. We also used a sensitive luciferase immunoprecipitation system (LIPS) assay to screen serum samples from these dogs for the presence of anti-CHV antibodies. Surprisingly, there was no evidence of exposure to, or a carrier state of, CHV in this large cohort, suggesting that the virus is not associated with CH in UK dogs. Future work, including transmission studies, is required to understand the pathogenesis of CHV in canids before it can be proposed as a surrogate model for HCV-induced liver disease in man.

Independence of herpesvirus-induced T cell lymphoma from viral cyclin D homologue.

Cyclin D family members are cellular protooncogenes, and their viral homologues in the Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus type 8 [HHV-8]) and the closely related Herpesvirus saimiri have been implicated as putative cofactors of viral transformation and pathogenesis. KSHV is regularly found in Kaposi's sarcoma and in the primary effusion B cell lymphoma and Castleman's disease associated with immunosuppression and AIDS. H. saimiri strain C488 transforms human and marmoset T cells in vitro and causes polyclonal T cell lymphoma in New World monkeys. The viral cyclins stimulate cell cycle progression of quiescent fibroblasts, and they form active cyclin-dependent kinase (CDK)6 complexes of broad substrate specificity that can resist and downregulate cellular CDK inhibitors. This study shows that the viral cyclin of H. saimiri strain C488 is not required for viral replication, T cell transformation, and pathogenicity in New World primates.

Major histocompatibility complex class I-associated vaccine protection from simian immunodeficiency virus-infected peripheral blood cells.

To evaluate the effectiveness of vaccine protection from infected cells from another individual of the same species, vaccinated rhesus macaques (Macaca mulatta) were challenged with peripheral blood mononuclear cells from another animal diagnosed with acquired immune deficiency syndrome (AIDS). Half of the simian immunodeficiency virus (SIV)-vaccinated animals challenged were protected, whereas unprotected vaccinates progressed as rapidly to AIDS. Protection was unrelated to either total antibody titers to human cells, used in the production of the vaccine, to HLA antibodies or to virus neutralizing activity. However, analysis of the serotype of each animal revealed that all animals protected against cell-associated virus challenge were those which were SIV vaccinated and which shared a particular major histocompatibility complex (MHC) class I allele (Mamu-A26) with the donor of the infected cells. Cytotoxic T lymphocytes (CTL) specific for SIV envelope protein were detected in three of four protected animals vs. one of four unprotected animals, suggesting a possible role of MHC class I-restricted CTL in protection from infected blood cells. These findings have possible implications for the design of vaccines for intracellular pathogens such as human immunodeficiency virus (HIV).

Detection of breastmilk antibodies targeting SARS-CoV-2 nucleocapsid, spike and receptor-binding-domain antigens.

A 40-year-old female was found to have strongly neutralizing SARS-CoV-2 breastmilk IgA and IgG antibodies reactive against multiple SARS-CoV-2 antigens at 2.5 months after documented infection with SARS-CoV-2. At 6.5 months following the infection, she remained positive for breastmilk and serum SARS-CoV-2 neutralizing antibodies. Holder breast milk pasteurization did not diminish SARS-CoV-2 antibody titres but it reduced its neutralizing capacity, while serum heat inactivation had no negative effect on SARS-CoV-2 serum antibody levels and neutralizing capacity. Current data on SARS-CoV-2 and breastmilk are reviewed.

Decreased expression of IL-2 in central and effector CD4 memory cells during progression to AIDS in rhesus macaques.

OBJECTIVE: HIV-1 infection in humans has been reported to lead to a shift in the cytokine balance, with a relative decrease in T helper 1 type cytokines, especially IL-2. On the basis of the expression of CD45RA, in combination with homing markers CD62L or alpha4beta7, T helper cells can be sub-divided into naive, activated naive, central memory and effector memory cells as well as gut-homing subpopulations. In addition, each subset may have the potential to express distinct cytokines. At present it is unclear whether the changes in cytokine expression observed in HIV-1-infected individuals are secondary to changes within the composition of CD4 T cell subsets or are caused by changes in cytokine expression within each subset. MATERIALS AND METHODS: A new technique was developed to detect cytokine expression in phorbol 12-myristate 13-acetate/ionomycin-activated CD62L and alpha4beta7-expressing CD4 T cell subsets, using the protease inhibitor KD-IX-73-4. RESULTS: In SIV-infected macaques that develop AIDS a marked decrease in IL-2 expression was found within central, effector, or gut-homing memory cell subsets, whereas the expression of IL-2 in naive T cell subsets remained unaffected. This reduced IL-2 expression by memory cells and not a loss of the frequency of CD4 memory cells accounted for the reduced expression of IL-2 by CD4 T cells during SIV infection. CONCLUSION: As defined by the cell surface markers utilized, it appears that progression to AIDS is associated with functional impairment of memory cells, but not changes in lymphocyte circulation patterns.

Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected with attenuated SIV: consequences of challenge.

OBJECTIVES: To determine whether prior infection with simian immunodeficiency virus (SIV)BK28 protects macaques from subsequent exposure to an HIV-1 envelope chimeric SIV (SHIV). Also, to determine the consequences of viral challenge on CD4 numbers and virus load on the current SIV infection. DESIGN AND METHODS: A total of 12 mature outbred Macacca mulatta were studied. Four naive controls and four previously infected with attenuated SIVBK28 were challenged with SHIV; four naive controls were not infected with SHIV. Sampling occurred twice monthly, and monthly thereafter. Changes in virus load, CD4 and CD8 populations were monitored. Highly sensitive and specific discriminative polymerase chain reaction (PCR) assays were used to distinguish between virus populations. RESULTS: SHIV readily infected challenged control animals, which developed a peak in virus load and a decline in CD4+ cell numbers. In controls, viral load declined and CD4 cell numbers rose to near normal levels after seroconversion. In contrast, in SIV-infected animals there was only a minor increase in viral load in only two out of four animals, 100-1000-fold lower than in naive animals. Interestingly, a decline in CD4 cells occurred in all four SIV-infected animals after SHIV challenge, which appeared more pronounced than in animals infected by SHIV alone. One SIV-infected animal which had low CD4 cell numbers at the time of SHIV challenge, developed a further decline in CD4 cells with a rising viral load. Discriminative PCR did not reveal SHIV in the challenged SIV animals. Interestingly the increase in viral load was due to SIV and not SHIV. CONCLUSIONS: Broad protection of animals previously infected with live attenuated SIV was demonstrated with protection from subsequent infection with HIV-1 envelope-bearing chimeric SIV. Subsequent exposure in cases with low CD4 cell numbers reveal the possibility of activation of the vaccine strain with the possible risk of inducing disease progression.

A clinically relevant HIV-1 subunit vaccine protects rhesus macaques from in vivo passaged simian-human immunodeficiency virus infection.

OBJECTIVES: To investigate whether immunization with recombinant HIV-1 envelope protein derived from a clinical isolate could protect macaques from infection with an in vivo passaged chimeric simian-human immunodeficiency virus (SHIV). DESIGN AND METHODS: A total of 16 animals were studied from which three groups of four animals were immunized with vaccine formulations of the CC-chemokine receptor-5-binding recombinant gp120 of HIV-1W6.1D. Four weeks after the last immunization, all 16 animals were intravenously challenged with in vivo passaged SHIV derived from the same HIV-1 group B clinical isolate (W6.1D) as the vaccines. RESULTS: Vaccine protection from infection was demonstrated in 10 out of 12 macaques immunized with recombinant gp120. Complete protection from infection was achieved with all of the animals that received the SBAS2-W6.1D formulation, a potent inducer of both T-cell and humoral immune responses. Partial protection was achieved with SBAS1-W6.1D, a formulation based on immunomodulators known to induce T-cell responses in humans. In vaccinated animals that were infected, virus load was reduced and infection was delayed. CONCLUSIONS: In a relatively large number of primates, vaccine efficacy was demonstrated with a clinically relevant HIV-1 vaccine. These results reveal that it is possible to induce sterilizing immunity sufficient to protect from infection with SHIV which was passaged multiple times in vivo. Our findings have implications for current HIV-1 clinical vaccine trials and ongoing efforts to develop safe prophylactic AIDS vaccines.

Direct amplification and cloning of up to 5-kb lentivirus genomes from serum.

To produce large cDNA strands from biological samples containing limited numbers of template molecules, it may be necessary to minimize both nonspecific primer attachment in first-strand synthesis and secondary structure in RNA molecules. Failure to do so could result in the accumulation of shortened cDNA strands and therefore may reduce the yield of large cDNA molecules, sometimes below detection level. We show that 5.0-kb cDNA fragments can be generated from simian immunodeficiency virus RNA in a specific reverse transcription (RT)-PCR by increasing the stringency of the primer-annealing conditions, followed by the elimination of excess free primer. Since this method utilizes a relatively long primer in the first-strand cDNA synthesis, it is possible to heat-denature the nonspecific RNA/primer complexes and RNA secondary structure without dissociating the primer from the specific template. In contrast to classic RT assays, in which an excess of primer is annealed to denatured RNA just prior to and during reverse transcription at relative low temperatures (37 degrees-42 degrees C), this method eliminates false priming. To optimize the yield and fidelity of full-length cDNA molecules, two PCR amplifications are first performed using both Taq and Pfu polymerase, followed by Pfu alone in the second amplification.

Comparative features of a coronavirus isolated from a cheetah with feline infectious peritonitis.

A coronavirus which was isolated from a cheetah (Acinonyx jubatus) that succumbed to feline infectious peritonitis was characterized in vitro. The virus was determined to be highly cell-associated with Crandell feline kidney (CrFK) cells and was routinely maintained as a persistent infection (CrFK 83-4497). The cheetah coronavirus was compared with other members of the feline coronavirus group including the feline enteric coronavirus (FECV) 79-1683 and the feline infectious peritonitis viruses (FIPV), 79-1146, and UCD-1. The cheetah coronavirus was demonstrated to have a restricted host-cell range with limited cytopathic effect. Indirect immunofluorescence with antisera to FIPV UCD-1 revealed the concentration of viral antigens in the perinuclear region of cells infected with the cheetah coronavirus. Ultrastructural studies of the cheetah coronavirus indicated a limited number of immature viral particles within cytoplasmic vesicles and at the cell surface. This was in contrast to electron microscopy results of FECV 79-1683 and FIPV 79-1146, which had numerous mature virus particles within the cytoplasmic vesicles, as well as at the cell surface. The cheetah coronavirus was tentatively placed in the feline coronavirus family based upon its antigenic reactivity by immunofluorescence; however, the possibility that it represents a unique coronavirus of cheetahs should not be dismissed without further analyses at the host and genomic levels.

The role of major histocompatibility complex polymorphisms on SIV infection in rhesus macaques.

To investigate whether Major Histocompatibility Complex (MHC) polymorphisms influence either susceptibility to SIV infection or progress to actual disease, rhesus monkeys were subjected to various forms of SIV infection and screened for allelic MHC heterogeneity by means of serological and biochemical methods. Animals that are protected against cell associated virus challenges were those that are SIV vaccinated and which shared a particular MHC class I allele (Mamu-A26) with the donor of the infected cells. Comparisons on the rate of infection to AIDS in SIVmac infected macaques showed that most Mamu-A26 positive animals belong to the group of long time survivors. In our outbred colony, about 25% of the rhesus macaques are positive for the Mamu-A26 serotype. Gel electrophoretic analyses demonstrated that isoelectric point (pI) differences of MHC class I heavy chains correlate with allotyping. In addition, the Mamu-A26 specificity was found to display heterogeneity. These results suggest that particular Mamu-A26 (associated) gene products may have the capacity or quality to induce antigen specific cytotoxic T lymphocyte responses that play a key role in controlling SIV infection or vaccine protection.

Rhesus macaque and chimpanzee DC-SIGN act as HIV/SIV gp120 trans-receptors, similar to human DC-SIGN.

Dendritic cells (DC) have been implicated in the pathogenesis of both human and simian immunodeficiency viruses (HIV and SIV, respectively). The DC-specific HIV-1 trans-receptor DC-SIGN is thought to be essential for viral dissemination by DC. Abundant expression in lymphoid tissues also implies a function for DC-SIGN in chronic HIV-1 infections, in facilitating persistent infection of T cells. We have therefore isolated the rhesus macaque and chimpanzee homologues of DC-SIGN to investigate their function in a primate model. Both rhesus macaque and chimpanzee DC-SIGN are highly similar to the human homologue. Three monoclonal antibodies against human DC-SIGN, AZN-D1, -D2 and -D3, cross-react with rhesus macaque DC-SIGN, whereas AZN-D2 does not cross-react with chimpanzee DC-SIGN. The primate homologues are abundantly expressed in lymphoid tissues such as lymph nodes, as well as in mucosal tissues involved in sexual transmission of HIV-1, and are functionally similar to human DC-SIGN. They have a high affinity for the immunological ligands of DC-SIGN: ICAM-2 and -3. Moreover, both homologues bind the HIV-1 envelope glycoprotein gp120 and therefore can act as a HIV-1 trans-receptor in the same way as human DC-SIGN. These data demonstrate that primate models are suitable to further dissect the role of DC-SIGN in the transmission and pathogenesis of infection with immunodeficiency viruses.

Changes in dendritic cell subsets in the lymph nodes of rhesus macaques after application of glucocorticoids.

Corticosteroids are used therapeutically as potent immunosuppressive and antiinflammatory drugs for a broad spectrum of diseases. Although corticosteroids are known to inhibit the production of many cytokines in activated T cells, there is also evidence for increases in IL-4 and in some cases IFNgamma production. These conflicting results may be caused by contrary effects of corticosteroids on different cell types involved in immune regulation, for instance antigen presenting cells (APC) versus T cells. In the present study we simultaneously investigated the effect of local as well as systemic application of glucocorticoids (GCC) on the phenotype of APC in the skin as well as the lymph nodes in a model primate species, the rhesus macaque. Using a range of APC markers, including CD68, HAM56, HLA-DR, CD1a, p55, RFD-1, and costimulatory molecules CD40, CD80, and CD86 we document the close phenotypic resemblance of rhesus and human APC. We noted that topical GCC treatment specifically lead to a marked decrease in the number of CD1a expressing cells in the draining lymph nodes. However, the number of CD1a positive cells in peripheral lymph nodes was not affected by systemic GCC treatment. Importantly, by performing double staining of CD1a with RFD-1 we observed a shift in the expression pattern of these dendritic cell markers in the lymph nodes, with an increase in the number of RFD-1 single positive cells relative to CD1a single positive and CD1a/RFD-1 double positive cells. These findings suggest that GCC treatment results in the presence of phenotypically more mature APC.

Inhibition of apoptosis in T cells expressing human T cell leukemia virus type I Tax.

This study set out to determine whether T cell dysfunction associated with HTLV-I led to increased sensitivity of infected cells to apoptosis or, owing to their potential to develop ATL, if infected cells would become resistant to this process. To test this hypothesis we utilized the monoclonal antibody anti-APO-1, which has been demonstrated to induce apoptosis in human T cells. Human T cell lines expressing HTLV-I showed reduced susceptibility to anti-APO-1-induced apoptosis despite expression of high levels of cell surface APO-1. Cell-free supernatant of the Tax-expressing cell line C8166 and heat-inactivated supernatant of the HTLV-I-producing cell line MT2 transferred increased resistance to anti-APO-1 to susceptible Jurkat T cells. Susceptible T cells transfected with an HTLV-I Tax-expressing vector or treated with soluble Tax protein became less susceptible to anti-APO-1-induced cell death. Furthermore, primary human lymphocytes treated with soluble Tax were less susceptible to apoptosis induced by anti-APO-1. The protective effect of Tax in T cell lines and primary human lymphocytes was reversed by the addition of anti-Tax antibodies. Anti-APO-1-induced apoptosis was also found to be inhibited in Jurkat cells by the induction of protein kinase C (PKC) with 12-O-tetradecanoylphorbol-13-acetate (TPA). Resistance to apoptosis conferred by HTLV-I Tax and an active PKC pathway may be factors contributing to the survival of dysregulated HTLV-I-infected T cells prone to the development of adult T cell leukemia.

The relative resistance of HIV type 1-infected chimpanzees to AIDS correlates with the maintenance of follicular architecture and the absence of infiltration by CD8+ cytotoxic T lymphocytes.

Lymphoid tissues are the focus of critical events in HIV pathogenesis. Persistent and high levels of virus production, extensive trapping of virus particles in germinal centers, and progressive degenerative changes in lymph node architecture are characteristics of progressive HIV-1 infection. Infiltrates of granzyme B- and TIA-expressing CD8+ "cytotoxic" T lymphocytes (CTLs) precede involution of germinal centers in humans who develop AIDS. Similar to humans, HIV-1 infection in chimpanzees is active and persistent. However, in contrast to humans, they remain relatively resistant to AIDS. Lymph node biopsies from chimpanzees infected with HIV-1 or a related chimpanzee lentivirus were studied for the level and pattern of virus expression, changes in lymphoid architecture, CD8+ T cell infiltrates and the presence or absence of CTL markers. In stark contrast to HIV-1-infected humans, lymph nodes from infected chimpanzees had little virus deposition in germinal centers and a paucity of virus-expressing cells. Although some of the lymph nodes examined from infected animals had moderate follicular hyperplasia with infiltrating CD8+ T cells, none had evidence of follicular fragmentation. Most importantly, in marked contrast to infected humans, CD8+ T cells infiltrating the germinal center were negative for the CTL marker granzyme B. This evidence suggests that the infiltration of CD8+ CTLs into the germinal centers of lymph nodes may be a key determinant in AIDS pathogenesis.

Vaccine protection and reduced virus load from heterologous macaque-propagated SIV challenge.

The efficacy of vaccine protection afforded by live attenuated vaccines was tested by heterologous SIVtno8980 challenge following successful protection against homologous SIVmac32H challenge. Animals immunized with the attenuated SIVmacBK28 molecular clone were asymptomatic and virus isolation negative by quantitative virus isolation prior to challenge. Two groups of four animals previously immunized 5 years and 4 months (respectively) were challenged with 100 MID50 of SIVtno8980, as was a third group of four naive controls. All control animals that were challenged developed high levels of plasma antigenemia within 2 weeks of challenge and developed rapid Th/m cell loss whereas vaccinated animals did not. Quantitative virus isolation from peripheral blood mononuclear cells revealed that one of four animals in each group became virus isolation positive but that the virus load in the two vaccinated animals was markedly lower than in nonvaccinated controls. Studies are underway to determine the duration and immunological correlates of protection from AIDS.

The effect of recombinant human interferon alpha B/D compared to interferon alpha 2b on SIV infection in rhesus macaques.

The model of simian immunodeficiency virus (SIV) infection in rhesus macaques was used to evaluate the effects of recombinant human interferon alpha, Hu IFN-alpha 2b and Hu IFN-gamma B,D, at two doses. Administration began 1 day prior to infection and was continued for 90 days postinfection. Both interferons suppressed SIV antigenemia during the treatment period. Following treatment animals were monitored for 4 years for rate of disease progression. Neither IFN prolonged the asymptomatic period or survival.

Detection of human T-cell leukaemia virus 1 permissive cells using cell lines producing selectable recombinant virions.

A selectable retrovirus vector based on a full length HTLV-1 provirus clone, pCS-HTLV-1, was constructed by replacing the coding regions for tax, rex and the 3' region of env with the prokaryotic neomycin resistance gene under the control of the CMV promoter. This vector, pHTLV-1-CMVneo, was transfected into HTLV-1 infected human lymphocytes and fibroblasts. The production of recombinant virus by these cells was measured by the transfer of G418 resistance to target cells. Infection of target cells showed a preference for human lymphocytes in addition to two human fibroblast cell lines, Hos7 and RD4, and the African green monkey kidney cell line, Cos7. This system provides a method to study the cellular tropism of HTLV-1 and additionally provides a model to facilitate molecular studies of the natural events of HTLV-1 infection and integration.

Cytochemical analysis of human T cell leukaemia virus I LTR-regulated beta-galactosidase gene expression using a novel integrated cell system.

To develop a reporter system to study the response of an integrated retroviral LTR and cellular and viral events which influence transcription, the 5' LTR of HTLV-1 was coupled to the Escherichia coli beta-galactosidase gene (lacZ). This construct was assembled within a vector containing the neomycin resistance gene controlled by the SV40 promoter, and introduced into HeLa cells. Expression from the LTR in one clone was upregulated by positive regulators of HTLV-1 expression, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and the HTLV-1 transregulatory protein (tax), as has been previously reported using transient transfection assays. This method proved to be a rapid and reproducible assay for the measurement of integrated viral LTR activation in a single cell system.