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.

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.

Seroprevalence of SV40-like polyomavirus infections in captive and free-ranging macaque species.

BACKGROUND AND METHODS: To investigate the seroprevalence of polyomavirus infections in macaques, we analyzed 1579 sera from nine different species for antibodies cross-reactive with simian virus 40 (SV40) in an enzyme-linked immunosorbent assay. Most samples were collected from captive animals, but we also investigated a colony of free-ranging Barbary macaques (Macaca sylvanus). RESULTS: High seropositive rates were found in rhesus macaques (Macaca mulatta; 74.7%), cynomolgus macaques (Macaca fascicularis; 44.8%) and Tonkean macaques (Macaca tonkeana; 41.7%), especially in animals imported from China. Low rates were measured in cynomolgus macaques from Mauritius (8.8%), and in Barbary macaques (1.4%). Seropositivity was age-dependent increasing to >70% in animals of 5 years and older. CONCLUSIONS: High seroprevalence rates were found in different species of macaques, dependent on their origin. Very low infection rates found in Barbary macaques and cynomolgus macaques from Mauritius suggest that these animals in the wild are not commonly infected by SV40-like viruses.

Comparison of intranasal with targeted lymph node immunization using PR8-Flu ISCOM adjuvanted HIV antigens in macaques.

The rapidly spreading HIV epidemic requires a vaccine that elicits potent mucosal immunity to halt or slow transmission. Induction of these responses will depend on the use of appropriate adjuvants and targeting of the mucosal immune system. Previously, immune stimulating complexes (ISCOM) have shown great potency as adjuvant in the induction of mucosal responses in mice and systemic responses in non-human primates. In this study, HIV formulated in PR8-Flu ISCOM adjuvant was applied to immunize rhesus macaques against HIV; targeting the mucosa either via intranasal (IN) application or via targeted lymph node immunization (TLNI). While, strong systemic, HIV specific, cytokine, lymphoproliferative, and antibody responses were induced via the TLNI route, the IN application generated only low responses. Furthermore, all four animals immunized via TLNI developed vaginal IgA antibodies against gp120. In conclusion, in contrast to what has been demonstrated in mice, the IN application of PR8-Flu ISCOM did not induce strong immune responses in rhesus macaques unlike those immunized by the TLNI route.

Zoonotic viral diseases and the frontier of early diagnosis, control and prevention.

Public awareness of the human health risks of zoonotic infections has grown in recent years. Currently, concern of H5N1 flu transmission from migratory bird populations has increased with foci of fatal human cases. This comes on the heels of other major zoonotic viral epidemics in the last decade. These include other acute emerging or re-emerging viral diseases such as severe acute respiratory syndrome (SARS), West-Nile virus, Ebola virus, monkeypox, as well as the more inapparent insidious slow viral and prion diseases. Virus infections with zoonotic potential can become serious killers once they are able to establish the necessary adaptations for efficient human-to-human transmission under circumstances sufficient to reach epidemic proportions. The monitoring and early diagnosis of these potential risks are overlapping frontiers of human and veterinary medicine. Here, current viral zoonotics and evolving threats are reviewed.

Experimental norovirus infections in non-human primates.

Noroviruses, with Norwalk virus as the prototype strain, are the most common cause of viral gastroenteritis in people of all ages. Limited information on the immunology of Norovirus infections has been obtained by studies both in the natural setting and in experimentally infected volunteers. Interpretation of these studies is difficult due to the lack of information on the history of Norovirus exposure and the cross-reactivity of antibodies. An animal model for Norovirus infections would be important to study the immune response, e.g., for vaccine assessment. In the present study the susceptibility of common marmosets, cotton top tamarins, cynomolgus, and rhesus macaques to Norovirus infection was tested. Following oral inoculation, low level replication may have occurred in common marmosets and cotton top tamarins but not in cynomolgus macaques, based on short-term viral shedding; neither clinical symptoms nor antibody responses were observed in these species. In contrast, rhesus macaques were found susceptible to Norwalk virus infection as one animal shed virus for a longer period of time and developed Norwalk virus specific IgM and IgG responses. Further research on Norovirus susceptibility in rhesus macaques may yield an animal model to study the immune response and pathogenesis after Norovirus infection.

CCR5 targeted SIV vaccination strategy preventing or inhibiting SIV infection.

Cell-surface CCR5 is a major coreceptor with CD4 glycoprotein, mediating cellular entry of CCR5 strains of HIV-1 or SIV. We targeted the SIV CCR5 coreceptor in a combined CCR5-SIV antigen immunization strategy. Rhesus macaques were immunized i.m. with the 70 kDa heat shock protein (HSP70) covalently linked to the CCR5 peptides, SIV gpl20 and p27. Intravenous challenge with SIV mac 8980 prevented SIV infection or decreased the viral load with the CCR5-SIV combined vaccine. CC chemokines and antibodies which block and downmodulateCCR5 were induced, as well as immune responses to the subunit SIV antigens. This novel vaccination strategy complements cognate immunity to SIV with innate immunity to the CCR5 coreceptor of SIV.

Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen.

The immunogenicity and protective efficacy of a DNA and recombinant modified vaccinia Ankara (MVA) vaccine administered by two different routes were investigated. DNA expressing HIV-1 IIIB env, gag, RT, rev, tat and nef, and MVA expressing HIV-1 IIIB nef, tat and rev and simian immunodeficiency virus (SIV) macJ5 gag/pol and vaccinia HIV-1 env, were used as immunogens. Four cynomolgus macaques received DNA intramuscularly (i.m.) at month 0 and intrarectally (i.r.) and intra-orally (i.o.) at 2 months, followed by MVA i.m. at 4 months and i.r. and i.o. at 8 months. Another group of four monkeys received the same immunogens but only i.m. Overall, stronger cellular immune responses measured by ELISPOT and T-cell proliferation assay were detected in the group primed i.m. and boosted mucosally. Following homologous intravenous simian-human immunodeficiency virus (SHIV) challenge, one of eight vaccinated animals was completely protected. This monkey, immunized i.m. and i.r.+i.o., exhibited the highest levels of HIV Env, Nef and Tat antibodies, high HIV Tat cytotoxic T-lymphocyte activity and T-lymphocyte proliferative responses to HIV Env. Four weeks post-challenge none of the monkeys immunized i.m. and i.r.+i.o., and only two out of four animals immunized i.m., demonstrated detectable plasma viral RNA levels. In contrast, all eight control animals had demonstrable plasma viral RNA levels 4 weeks post-challenge. Thus, stronger cellular immune responses and reduction of challenge virus burden were demonstrated in animals immunized i.m. as well as mucosally, compared with animals immunized i.m. only. The breadth and magnitude of the induced immune responses correlated with protective efficacy.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Gene therapy in nonhuman primate models of human autoimmune disease.

Before autoimmune diseases in humans can be treated with gene therapy, the safety and efficacy of the used vectors must be tested in valid experimental models. Monkeys, such as the rhesus macaque or the common marmoset, provide such models. This publication reviews the state of the art in monkey models for rheumatoid arthritis and multiple sclerosis and the (few) gene therapy experiments that have been performed in these models.

Reduced transmission and prevalence of simian T-cell lymphotropic virus in a closed breeding colony of chimpanzees (Pan troglodytes verus).

A retrospective study spanning 20 years was undertaken to investigate the prevalence and modes of transmission of a simian T-cell lymphotropic virus (STLV) in a closed breeding colony of chimpanzees. Of the 197 animals tested, 22 had antibodies that were cross-reactive with human T-cell lymphotropic virus type-1 (HTLV-I) antigens. The specificity of the antibody response was confirmed by Western blot analysis and the presence of a persistent virus infection was established by PCR analysis of DNA from peripheral blood mononuclear cells. Sequence analysis revealed that the virus infecting these chimpanzees was not HTLV-I but STLV(cpz), a virus that naturally infects chimpanzees. The limited number of transmission events suggested that management practices of social housing of family units away from troops of mature males might have prevented the majority of cases of transmission. Evidence for transmission by blood-to-blood contact was documented clearly in at least one instance. In contrast, transmission from infected mother to child was not observed, suggesting that this is not a common route of transmission for STLV in this species, which is in contrast to HTLV-1 in humans.

Genetic variability of the V1 and V2 env domains of SIVcpz-ant and neutralization pattern of plasma viruses in a chimpanzee infected naturally.

Specific neutralizing epitope changes have been observed in a chimpanzee infected naturally with SIVcpz, which differ from HIV-1 infecting humans. To characterize further these changes, a longitudinal study of env genomic sequence variation of SIVcpz-ant isolates was undertaken in this animal. The V1 and V2 regions of the env were determined to arise from specific recombination events. To determine whether recombination of the V1 and V2 domains was possibly associated with the emergence of neutralization escape viruses, envelope sequences and gene length polymorphisms from PBMC and plasma viral variants were studied over a 7-year period. PBMCs and plasma-associated infectious virus titers as well as plasma RNA viral loads were monitored longitudinally. The first 5 viruses isolated from the plasma were found to be neutralization escape variants. Sequence analysis of their V1 and the V2 regions indicated that a 20 amino acid stretch of the V1 region had undergone recombination and was also associated with the emergence of isolates eliciting strong neutralization responses. These findings support the hypothesis that recombination of the V1 and V2 regions of the envelope play a role in neutralization escape of SIVcpz in chimpanzees infected naturally. Furthermore, the data confirm that the neutralizing antibody response plays an important role in the decline of plasma infectious virus titers in HIV-1 related SIVcpz nonpathogenic infection.

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.

Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees.

OBJECTIVE: A comparative study of the replication kinetics of different HIV-1 variants (including SIV(cpz)) was undertaken to determine which viral characteristics were associated with sustained plasma viraemia in chimpanzees. DESIGN: Plasma samples from chimpanzees infected with six different HIV-1 clade B isolates were compared with plasma samples from SIV(cpz-ant)-infected chimpanzees. METHODS: A pan-clade quantitative competitive reverse transcriptase-polymerase chain reaction assay was developed based on conserved primer sequences recognizing M, N and O human lentiviruses as well as different SIV(cpz) isolates. RESULTS: Important differences between early kinetics in the human lentivirus isolates as well as compared with the chimpanzee isolate SIV(cpz-ant) were observed. R5-dependent non-syncytium-inducing (NSI) isolates (5016, Ba-L, SIV(cpz)) were found to have relatively higher viral loads than the syncytium-inducing (SI), X4-dependent primary (SF2), T cell-adapted (IIIB) or X4/R5 (Han2, DH12) SI primary isolates. CONCLUSION: Infection of chimpanzees with NSI R5-utilizing isolates correlated with persistent viraemia (approximately 10(4) RNA equivalents/ml) in contrast to transient viraemia observed after infection with SI X4-utilizing isolates.

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.

Rational development of prophylactic HIV vaccines based on structural and regulatory proteins.

The severity of the AIDS epidemic clearly emphasises the urgent need to expedite HIV vaccine candidates into clinical trials. Prophylactic HIV vaccine candidates have been evaluated in non-human primates. Based on specific proof of principle studies the first phase III clinical studies have recently begun in humans. However, a truly effective HIV vaccine is not yet at hand and many problems related to specific properties of the virus remain to be overcome. Previously proven empirical approaches have largely failed and now rational thinking based on an understanding of immunity to lentiviral infections is needed. This review addresses the scientific problems and complications facing the development of an HIV vaccine as well as the possible strategies currently available to overcome these problems. Recent attention has focussed on identifying the immune correlates and mechanisms of protection from either HIV infection or protection from disease progression. Based on these observations, the logic and rational behind the development of multiple component vaccine strategies are highlighted.

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys.

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.

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.