Evaluating the antibody response elicited by diverse HIV envelope immunogens in the African green monkey (Vervet) model.

African Green (Vervet) monkeys have been extensively studied to understand the pathogenesis of infectious diseases. Using vervet monkeys as pre-clinical models may be an attractive option for low-resourced areas as they are found abundantly and their maintenance is more cost-effective than bigger primates such as rhesus macaques. We assessed the feasibility of using vervet monkeys as animal models to examine the immunogenicity of HIV envelope trimer immunogens in pre-clinical testing. Three groups of vervet monkeys were subcutaneously immunized with either the BG505 SOSIP.664 trimer, a novel subtype C SOSIP.664 trimer, CAP255, or a combination of BG505, CAP255 and CAP256.SU SOSIP.664 trimers. All groups of vervet monkeys developed robust binding antibodies by the second immunization with the peak antibody response occurring after the third immunization. Similar to binding, antibody dependent cellular phagocytosis was also observed in all the monkeys. While all animals developed potent, heterologous Tier 1 neutralizing antibody responses, autologous neutralization was limited with only half of the animals in each group developing responses to their vaccine-matched pseudovirus. These data suggest that the vervet monkey model may yield distinct antibody responses compared to other models. Further study is required to further determine the utility of this model in HIV immunization studies.

The Road to Elimination: Current State of Schistosomiasis Research and Progress Towards the End Game.

The new WHO Roadmap for Neglected Tropical Diseases targets the global elimination of schistosomiasis as a public health problem. To date, control strategies have focused on effective diagnostics, mass drug administration, complementary and integrative public health interventions. Non-mammalian intermediate hosts and other vertebrates promote transmission of schistosomiasis and have been utilized as experimental model systems. Experimental animal models that recapitulate schistosomiasis immunology, disease progression, and pathology observed in humans are important in testing and validation of control interventions. We discuss the pivotal value of these models in contributing to elimination of schistosomiasis. Treatment of schistosomiasis relies heavily on mass drug administration of praziquantel whose efficacy is comprised due to re-infections and experimental systems have revealed the inability to kill juvenile schistosomes. In terms of diagnosis, nonhuman primate models have demonstrated the low sensitivity of the gold standard Kato Katz smear technique. Antibody assays are valuable tools for evaluating efficacy of candidate vaccines, and sera from graded infection experiments are useful for evaluating diagnostic sensitivity of different targets. Lastly, the presence of Schistosomes can compromise the efficacy of vaccines to other infectious diseases and its elimination will benefit control programs of the other diseases. As the focus moves towards schistosomiasis elimination, it will be critical to integrate treatment, diagnostics, novel research tools such as sequencing, improved understanding of disease pathogenesis and utilization of experimental models to assist with evaluating performance of new approaches.

Infection of Chinese Rhesus Monkeys with a Subtype C SHIV Resulted in Attenuated In Vivo Viral Replication Despite Successful Animal-to-Animal Serial Passages.

Rhesus macaques can be readily infected with chimeric simian-human immunodeficiency viruses (SHIV) as a suitable virus challenge system for testing the efficacy of HIV vaccines. Three Chinese-origin rhesus macaques (ChRM) were inoculated intravenously (IV) with SHIVC109P4 in a rapid serial in vivo passage. SHIV recovered from the peripheral blood of the final ChRM was used to generate a ChRM-adapted virus challenge stock. This stock was titrated for the intrarectal route (IR) in 8 ChRMs using undiluted, 1:10 or 1:100 dilutions, to determine a suitable dose for use in future vaccine efficacy testing via repeated low-dose IR challenges. All 11 ChRMs were successfully infected, reaching similar median peak viraemias at 1-2 weeks post inoculation but undetectable levels by 8 weeks post inoculation. T-cell responses were detected in all animals and Tier 1 neutralizing antibodies (Nab) developed in 10 of 11 infected ChRMs. All ChRMs remained healthy and maintained normal CD4+ T cell counts. Sequence analyses showed >98% amino acid identity between the original inoculum and virus recovered at peak viraemia indicating only minimal changes in the env gene. Thus, while replication is limited over time, our adapted SHIV can be used to test for protection of virus acquisition in ChRMs.

Chronic schistosomiasis suppresses HIV-specific responses to DNA-MVA and MVA-gp140 Env vaccine regimens despite antihelminthic treatment and increases helminth-associated pathology in a mouse model.

Future HIV vaccines are expected to induce effective Th1 cell-mediated and Env-specific antibody responses that are necessary to offer protective immunity to HIV infection. However, HIV infections are highly prevalent in helminth endemic areas. Helminth infections induce polarised Th2 responses that may impair HIV vaccine-generated Th1 responses. In this study, we tested if Schistosoma mansoni (Sm) infection altered immune responses to SAAVI candidate HIV vaccines (DNA and MVA) and an HIV-1 gp140 Env protein vaccine (gp140) and whether parasite elimination by chemotherapy or the presence of Sm eggs (SmE) in the absence of active infection influenced the immunogenicity of these vaccines. In addition, we evaluated helminth-associated pathology in DNA and MVA vaccination groups. Mice were chronically infected with Sm and vaccinated with DNA+MVA in a prime+boost combination or MVA+gp140 in concurrent combination regimens. Some Sm-infected mice were treated with praziquantel (PZQ) prior to vaccinations. Other mice were inoculated with SmE before receiving vaccinations. Unvaccinated mice without Sm infection or SmE inoculation served as controls. HIV responses were evaluated in the blood and spleen while Sm-associated pathology was evaluated in the livers. Sm-infected mice had significantly lower magnitudes of HIV-specific cellular responses after vaccination with DNA+MVA or MVA+gp140 compared to uninfected control mice. Similarly, gp140 Env-specific antibody responses were significantly lower in vaccinated Sm-infected mice compared to controls. Treatment with PZQ partially restored cellular but not humoral immune responses in vaccinated Sm-infected mice. Gp140 Env-specific antibody responses were attenuated in mice that were inoculated with SmE compared to controls. Lastly, Sm-infected mice that were vaccinated with DNA+MVA displayed exacerbated liver pathology as indicated by larger granulomas and increased hepatosplenomegaly when compared with unvaccinated Sm-infected mice. This study shows that chronic schistosomiasis attenuates both HIV-specific T-cell and antibody responses and parasite elimination by chemotherapy may partially restore cellular but not antibody immunity, with additional data suggesting that the presence of SmE retained in the tissues after antihelminthic therapy contributes to lack of full immune restoration. Our data further suggest that helminthiasis may compromise HIV vaccine safety. Overall, these findings suggested a potential negative impact on future HIV vaccinations by helminthiasis in endemic areas.

DNA-MVA-protein vaccination of rhesus macaques induces HIV-specific immunity in mucosal-associated lymph nodes and functional antibodies.

Successful future HIV vaccines are expected to generate an effective cellular and humoral response against the virus in both the peripheral blood and mucosal compartments. We previously reported the development of DNA-C and MVA-C vaccines based on HIV-1 subtype C and demonstrated their immunogenicity when given in a DNA prime-MVA boost combination in a nonhuman primate model. In the current study, rhesus macaques previously vaccinated with a DNA-C and MVA-C vaccine regimen were re-vaccinated 3.5years later with MVA-C followed by a protein vaccine based on HIV-1 subtype C envelope formulated with MF59 adjuvant (gp140Env/MF59), and finally a concurrent boost with both vaccines. A single MVA-C re-vaccination elicited T cell responses in all animals similar to previous peak responses, with 4/7 demonstrating responses >1000 SFU/106 PBMC. In contrast to an Env/MF59-only vaccine, concurrent boosting with MVA-C and Env/MF59 induced HIV-specific cellular responses in multiple mucosal associated lymph nodes in 6/7 animals, with high magnitude responses in some animals. Both vaccine regimens induced high titer Env-specific antibodies with ADCC activity, as well as neutralization of Tier 1 viruses and modest Tier 2 neutralization. These data demonstrate the feasibility of inducing HIV-specific immunity in the blood and mucosal sites of viral entry by means of DNA and poxvirus-vectored vaccines, in combination with a HIV envelope-based protein vaccine.

Transient global T cell activation after vaccination of rhesus macaques with a DNA-poxvirus vaccine regimen for HIV.

Persistent T cell activation following immunization with HIV vaccines may increase HIV acquisition risk. We investigated the magnitude and kinetics of T cell activation following vaccination of rhesus macaques with a candidate HIV vaccine consisting of a recombinant DNA and MVA vaccination regimen. We show that global CD4+ and CD8+ T cell activation, as measured by the expression of Ki67 and Bcl-2, peaked one week after boosting with MVA, but then waned rapidly to pre-vaccination levels. Furthermore, increased frequencies of CD4+ CCR5+ T cells, which represent potential HIV target cells, were short-lived and decreased to baseline levels within two months. Activated CD4+ T cells were predominantly of a central memory phenotype, and activated CD8+ T cells were distributed between central and effector memory phenotypes. Thus, only transient changes in T cell activation occurred following poxvirus vaccination, indicating a lack of persistent immune activation.

The novel capripoxvirus vector lumpy skin disease virus efficiently boosts modified vaccinia Ankara human immunodeficiency virus responses in rhesus macaques.

Poxvirus vectors represent promising human immunodeficiency virus (HIV) vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant capripoxvirus vector, lumpy skin disease virus (LSDV), in combination with modified vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrated that the combination regimen was immunogenic in rhesus macaques, inducing high-magnitude, broad and balanced CD4(+) and CD8(+) T-cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.

Robust immunity to an auxotrophic Mycobacterium bovis BCG-VLP prime-boost HIV vaccine candidate in a nonhuman primate model.

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55(gag) virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4(+) and CD8(+) T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

Measurements of immune responses for establishing correlates of vaccine protection against HIV.

Well-defined correlates of protective immunity are an essential component of rational vaccine development. Despite years of basic science and three HIV vaccine efficacy trials, correlates of immunological protection from HIV infection remain undefined. In December 2010, a meeting of scientists engaged in basic and translational work toward developing HIV-1 vaccines was convened. The goal of this meeting was to discuss current opportunities and optimal approaches for defining correlates of protection, both for ongoing and future HIV-1 vaccine candidates; specific efforts were made to engage young scientists. We discuss here the highlights from the meeting regarding the progress made and the way forward for a protective HIV-1 vaccine.

A prime-boost immunisation regimen using recombinant BCG and Pr55(gag) virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons.

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55(gag) virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55(gag) VLPs.

Broad, high-magnitude and multifunctional CD4+ and CD8+ T-cell responses elicited by a DNA and modified vaccinia Ankara vaccine containing human immunodeficiency virus type 1 subtype C genes in baboons.

Candidate human immunodeficiency virus (HIV) vaccine regimens based on DNA boosted with recombinant modified vaccinia Ankara (MVA) have been in development for some time, and there is evidence for improved immunogenicity of newly developed constructs. This study describes immune responses to candidate DNA and MVA vaccines expressing multiple genes (gag, RT, tat, nef and env) from HIV-1 subtype C in chacma baboons (Papio ursinus). The vaccine regimen induced (i) strong T-cell responses, with a median of 4103 spot forming units per 10(6) peripheral blood mononuclear cells by gamma interferon (IFN-gamma) ELISPOT, (ii) broad T-cell responses targeting all five vaccine-expressed genes, with a median of 12 peptides targeted per animal and without any single protein dominating the response, (iii) balanced CD4(+) and CD8(+) responses, which produced both IFN-gamma and interleukin (IL)-2, including IL-2-only responses not detected by the ELISPOT assay, (iv) vaccine memory, which persisted 1 year after immunization and could be boosted further, despite strong anti-vector responses, and (v) mucosal T-cell responses in iliac and mesenteric lymph nodes in two animals tested. The majority of peptide responses mapped contained epitopes previously identified in human HIV infection, and two high-avidity HIV epitope responses were confirmed, indicating the utility of the baboon model for immunogenicity testing. Together, our data show that a combination of DNA and MVA immunization induced robust, durable, multifunctional CD4(+) and CD8(+) responses in baboons targeting multiple HIV epitopes that may home to mucosal sites. These candidate vaccines, which are immunogenic in this pre-clinical model, represent an alternative to adenoviral-based vaccines and have been approved for clinical trials.

HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine.

A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 10(6) peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 10(6) PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 10(6) PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-gamma responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-gamma response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials.

Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-gamma ELISPOT tests following a tuberculosis outbreak.

BACKGROUND: Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB). METHODS: Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-gamma ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. RESULTS: Both ESAT-6 and CFP-10 IFN-gamma ELISPOT assays were able to detect early M. tuberculosis but also M. intracellulare infection. Although this indicates potential cross-reactivity with M. intracellulare antigens, the method was able to distinguish M. bovis BCG vaccination from M. tuberculosis infection. This assay performed better than the TST, which failed to detect one M. tuberculosis and two early M. intracellulare infections. CONCLUSION: These results suggest that the IFN-gamma ELISPOT assay could improve the detection of M tuberculosis infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with M. intracellulare.

Widely varying SIV prevalence rates in naturally infected primate species from Cameroon.

Although it is now well established that a substantial proportion of wild-living primates in sub-Saharan Africa harbor SIV, no study to date has examined to what extent the various species are naturally infected. In this study, we first describe the development and validation of sensitive and specific SIV antibody detection assays representing all major known primate lentiviral lineages on a panel of 207 sera from 11 different primate species with known infection status. The newly developed assays were then used to determine SIV prevalence rates in nine primate species native to Cameroon. Analysis of 722 sera revealed widely varying prevalence rates, ranging from an apparent absence of SIV infection in crested mona (0/70), grey cheeked (0/36) and agile mangabeys (0/92), to prevalence rates of 3%, 4%, 11%, 27%, 39% and 52% for mustached (6/203), greater spot-nosed (8/193), northern talapoin (3/26), mantled guereza (14/52), De Brazza's (9/23) and mandrill (14/27) monkeys, respectively. The epidemiology of naturally occurring SIV infections is thus more complex than previously appreciated and the various non-human primate hosts seem to differ in their susceptibility to SIV infection. The newly developed assays should now permit to define with greater accuracy existing SIV reservoirs and associated human zoonotic risk.

Experimental infection of non-human primates with a human rotavirus isolate.

Several rotavirus candidate vaccines have been developed and are at various stages of evaluation. In order to assess the safety and efficacy of these candidate vaccines, an appropriate non-human primate model is desirable. In earlier studies, we reported the presence of naturally occurring anti-rotavirus antibodies in monkeys and demonstrated that parenteral vaccination of baboons led to production of specific rotavirus antibodies in their milk. In the present study, we assessed the possibility of developing the baboon and the vervet monkey as an animal model for rotavirus studies by inoculating them with a pathogenic human rotavirus isolate prepared from the fresh faeces obtained from a child suffering from rotavirus diarrhoea. Preliminary studies have showed excretion of rotavirus in the faeces of 5 of 5 vervets monkeys and 1 of 2 baboons, by antigen ELISA and SDS-PAGE. These results were confirmed by RT-PCR and electron microscopy. The animals also showed elevation of IgG and high titres of virus neutralising antibodies. These data indicate that baboon and vervet monkeys may be useful models for human rotavirus infection and for pre-clinical evaluation of rotavirus candidate vaccines.

New simian immunodeficiency virus infecting De Brazza's monkeys (Cercopithecus neglectus): evidence for a cercopithecus monkey virus clade.

Nearly complete sequences of simian immunodeficiency viruses (SIVs) infecting 18 different nonhuman primate species in sub-Saharan Africa have now been reported; yet, our understanding of the origins, evolutionary history, and geographic distribution of these viruses still remains fragmentary. Here, we report the molecular characterization of a lentivirus (SIVdeb) naturally infecting De Brazza's monkeys (Cercopithecus neglectus). Complete SIVdeb genomes (9,158 and 9227 bp in length) were amplified from uncultured blood mononuclear cell DNA of two wild-caught De Brazza's monkeys from Cameroon. In addition, partial pol sequences (650 bp) were amplified from four offspring of De Brazza's monkeys originally caught in the wild in Uganda. Full-length (9068 bp) and partial pol (650 bp) SIVsyk sequences were also amplified from Sykes's monkeys (Cercopithecus albogularis) from Kenya. Analysis of these sequences identified a new SIV clade (SIVdeb), which differed from previously characterized SIVs at 40 to 50% of sites in Pol protein sequences. The viruses most closely related to SIVdeb were SIVsyk and members of the SIVgsn/SIVmus/SIVmon group of viruses infecting greater spot-nosed monkeys (Cercopithecus nictitans), mustached monkeys (Cercopithecus cephus), and mona monkeys (Cercopithecus mona), respectively. In phylogenetic trees of concatenated protein sequences, SIVdeb, SIVsyk, and SIVgsn/SIVmus/SIVmon clustered together, and this relationship was highly significant in all major coding regions. Members of this virus group also shared the same number of cysteine residues in their extracellular envelope glycoprotein and a high-affinity AIP1 binding site (YPD/SL) in their p6 Gag protein, as well as a unique transactivation response element in their viral long terminal repeat; however, SIVdeb and SIVsyk, unlike SIVgsn, SIVmon, and SIVmus, did not encode a vpu gene. These data indicate that De Brazza's monkeys are naturally infected with SIVdeb, that this infection is prevalent in different areas of the species' habitat, and that geographically diverse SIVdeb strains cluster in a single virus group. The consistent clustering of SIVdeb with SIVsyk and the SIVmon/SIVmus/SIVgsn group also suggests that these viruses have evolved from a common ancestor that likely infected a Cercopithecus host in the distant past. The vpu gene appears to have been acquired by a subset of these Cercopithecus viruses after the divergence of SIVdeb and SIVsyk.

Prevalence of rotavirus antibodies in a non-human primate colony.

A total of 51 monkeys maintained in a colony at the Institute of Primate Research (Kenya) and housed in doors with natural lighting in a group cage were used in this study. Monkeys belonging to 3 species were selected at random and blood samples collected. The serum samples were screened for presence of neutralizing antibodies (VTN) to rhesus rotavirus (RRV) by virus neutralization assay. Virus neutralization was determined by 60% reduction in fluorescent focus units (ffu). 96% of the animals screened had naturally occurring antibodies to rhesus rotavirus. Another group of 11 lactating monkeys (5 baboons, 6 vervets) and their infants were screened further for presence of IgG and IgA antibodies in serum and breast milk (mothers). Overall, the mothers had higher titres of both IgG and IgA than the infants. Taken together, these results demonstrate rotavirus infection is endemic in this primate colony. This mimics the human situation, hence, captive non human primates (such as the baboons) could be a suitable model for testing rotavirus candidate vaccines and for investigating the possible application in humans of passive-active immunization strategy.