Isolation and biological characterization of an adenovirus of rhesus macaques.

The etiology of a disease in rhesus monkeys the main clinical manifestation of which was acute conjunctivitis of an epizootic character has been studied. The cytopathogenic agent well propagating in primarily trypsinized kidney cells of monkeys has been isolated when investigating the affected eye mucosa. It was not pathogenic for laboratory animals. The mean diameter of the virions is 75 nm, the buoyant density in CsCl is 1.34 g/cm3, the viral DNA density is 1.706 g/cm3. The biological properties and findings of physicochemical, electron-microscopic, and serologic investigations allow one to allocate the isolated agent to the SV-37 strain, a representative of the adenovirus group.     

Exploratory analysis of the effect of helminth infection on the immunogenicity and efficacy of the asexual blood-stage malaria vaccine candidate GMZ2

BackgroundHelminths can modulate the host immune response to Plasmodium falciparum and can therefore affect the risk of clinical malaria. We assessed here the effect of helminth infections on both the immunogenicity and efficacy of the GMZ2 malaria vaccine candidate, a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of P. falciparum. Controlled human malaria infection (CHMI) was used to assess the efficacy of the vaccine.MethodologyIn a randomized, double-blind Phase I clinical trial, fifty, healthy, lifelong malaria-exposed adult volunteers received three doses of GMZ2 adjuvanted with either Cationic Adjuvant Formulation (CAF) 01 or Alhydrogel, or a control vaccine (Rabies) on days (D) 0, D28 and D56, followed by direct venous inoculation (DVI) of 3,200 P. falciparum sporozoites (PfSPZ Challenge) approximately 13 weeks after last vaccination to assess vaccine efficacy. Participants were followed-up on a daily basis with clinical examinations and thick blood smears to monitor P. falciparum parasitemia for 35 days. Malaria was defined as the presence of P. falciparum parasites in the blood associated with at least one symptom that can be associated to malaria over 35 days following DVI of PfSPZ Challenge. Soil-transmitted helminth (STH) infection was assessed by microscopy and by polymerase chain reaction (PCR) on stool, and Schistosoma infection was assessed by microscopy on urine. Participants were considered as infected if positive for any helminth either by PCR and/or microscopy at D0 and/or at D84 (Helm+) and were classified as mono-infection or co-infection. Total vaccine-specific IgG concentrations assessed on D84 were analysed as immunogenicity outcome.Main findingsThe helminth in mono-infection, particularly Schistosoma haematobium and STH were significantly associated with earlier malaria episodes following CHMI, while no association was found in case of coinfection. In further analyses, the anti-GMZ2 IgG concentration on D84 was significantly higher in the S. haematobium-infected and significantly lower in the Strongyloides stercoralis-infected groups, compared to helminth-negative volunteers. Interesting, in the absence of helminth infection, a high anti-GMZ2 IgG concentration on D84 was significantly associated with protection against malaria.ConclusionsOur results suggest that helminth infection may reduce naturally acquired and vaccine-induced protection against malaria. Vaccine-specific antibody concentrations on D84 may be associated with protection in participants with no helminth infection. These results suggest that helminth infection affect malaria vaccine immunogenicity and efficacy in helminth endemic countries.     

Reduction of peak viremia by an integration-defective SIV proviral DNA vaccine in rhesus macaques

An integrase-defective SIV (idSIV) vaccine delivered by a DNA prime and viral particle boost approach can suppress viral loads (VLs) during the acute infection stage after intravenous SIVmac239 challenge. This study investigated how idSIV DNA and viral particle immunization alone contributed to the suppression of VLs in Chinese rhesus macaques after SIV challenge. Two macaques were immunized with idSIV DNA five times and two macaques were immunized with idSIV viral particles three times. Cellular and humoral immune responses were measured in the vaccinated macaques after immunization. The VLs and CD4⁺ T cell counts were monitored for 28 weeks after the intravenous SIVmac239 challenge. The SIV-specific T cell responses were only detected in the DNA-vaccinated macaques. However, binding and neutralizing antibodies against autologous and heterologous viruses were moderately better in macaques immunized with viral particles than in macaques immunized with DNA. After the challenge, the mean peak viremia in the DNA group was 2.3 logs lower than that in the control group, while they were similar between the viral particle immunization and control groups. Similar CD4⁺ T cell counts were observed among all groups. These results suggest that idSIV DNA immunization alone reduces VLs during acute infection after SIV challenge in macaques and may serve as a key component in combination with other immunogens as prophylactic vaccines.     

Accelerated heterologous adenovirus prime-boost SIV vaccine in neonatal rhesus monkeys

A pediatric human immunodeficiency virus type 1 (HIV-1) vaccine would be desirable to protect infants against HIV-1 transmission from breast-feeding. Such a vaccine would need to induce protective immunity at mucosal surfaces in neonates as soon as possible after birth. Recombinant adenovirus (rAd) vectors have been shown to elicit potent systemic and mucosal virus-specific immune responses in adult nonhuman primates and humans, but these vectors have not previously been comprehensively studied in infants. In this study, we demonstrate that a single injection of rAd26 encoding simian immunodeficiency virus mac239 (SIVmac239) Gag on the day of birth elicited detectable Gag-specific cellular immune responses in rhesus monkeys, but these responses were transient and waned quickly. In contrast, an accelerated heterologous prime-boost regimen involving administration of rAd35 at birth and rAd26 at 4 weeks of life elicited potent and durable Gag-specific cellular and humoral immune responses in neonatal rhesus monkeys, including mucosal responses that remained detectable at 1 year of age. These results suggest the potential of an accelerated heterologous rAd prime-boost regimen as a candidate HIV-1 vaccine for newborns.     

Periodicity and synchronization in blood-stage malaria infection

Malaria fever is highly periodic and is associated with the parasite replication cycles in red blood cells. The existence of periodicity in malaria infection demonstrates that parasite replication in different red blood cells is synchronized. In this article, rigorous mathematical analysis of an age-structured human malaria model of infected red blood cells (Rouzine and McKenzie, Proc Natl Acad Sci USA 100:3473–3478, 2003) is provided and the synchronization of Plasmodium falciparum erythrocytic stages is investigated. By using the replication rate as the bifurcation parameter, the existence of Hopf bifurcation in the age-structured malaria infection model is obtained. Numerical simulations indicate that synchronization with regular periodic oscillations (of period 48 h) occurs when the replication rate increases. Therefore, Kwiatkowski and Nowak’s observation (Proc Natl Acad Sci USA 88:5111–5113, 1991) that synchronization could be generated at modest replication rates is confirmed.     

恶性疟原虫抗原表位的亚单位疫苗与DNA疫苗的联合免疫

构建了含有恶性疟原虫抗原基因 ( AWTE)的真核表达质粒 p CMV- AWTE,以及能在大肠杆菌中得到分泌性表达的原核表达质粒 p MC0 5 ,表达的蛋白 AWTE保持了疟原虫抗原的抗原性。将 p CMV- AWTE以及 AWTE两者混合各 1 0μg鼻腔免疫小鼠 ,一次后诱导机体产生了较高水平的体液免疫及细胞免疫     

Immunogenicity and protective efficacy of DNA vaccines expressing rhesus cytomegalovirus glycoprotein B, phosphoprotein 65-2, and viral interleukin-10 in rhesus macaques

Rhesus cytomegalovirus (RhCMV) infection of macaques exhibits strong similarities to human CMV (HCMV) persistence and pathogenesis. The immunogenicity of DNA vaccines encoding three RhCMV proteins (a truncated version of glycoprotein B lacking the transmembrane region and endodomain [gBDeltaTM], phosphoprotein 65-2 [pp65-2], and viral interleukin-10 [vIL-10]) was evaluated in rhesus macaques. Two groups of monkeys (four per group) were genetically immunized four times with a mixture of either pp65-2 and gBDeltaTM or pp65-2, vIL-10, and gBDeltaTM. The vaccinees developed anti-gB and anti-pp65-2 antibodies in addition to pp65-2 cellular responses after the second booster immunization, with rapid responses observed with subsequent DNA injections. Weak vIL-10 immune responses were detected in two of the four immunized animals. Neutralizing antibodies were detected in seven monkeys, although titers were weak compared to those observed in naturally infected animals. The immunized monkeys and na?ve controls were challenged intravenously with 10(5) PFU of RhCMV. Anamnestic binding and neutralizing antibody responses were observed 1 week postchallenge in the vaccinees. DNA vaccination-induced immune responses significantly decreased peak viral loads in the immunized animals compared to those in the controls. No difference in peak viral loads was observed between the pp65-2/gBDeltaTM DNA- and pp65-2/vIL-10/gBDeltaTM-vaccinated groups. Antibody responses to nonvaccine antigens were lower postchallenge in both vaccine groups than in the controls, suggesting long-term control of RhCMV protein expression. These data demonstrated that DNA vaccines targeting the RhCMV homologues of HCMV gB and pp65 altered the course of acute and persistent RhCMV infection in a primate host.     

Antibody responses to two encephalomyocarditis virus vaccines in rhesus macaques (Macaca mulatta)

Abstract: Two groups of rhesus macaques (Macaca mulatta) housed in rodent-controlled outdoor corrals were inoculated with two different encephalomyocarditis virus (EMCV) vaccines. One group (n = 45) received a vaccine made from an inactivated field isolate of virus cultured during an outbreak at a zoo in Florida. This vaccine produced fourfold increases in the titers of 28 animals (62%); the increases persisted for at least 18 months (last test) after a single injection of the vaccine. The other group (n = 51) received a vaccine made from an inactivated porcine field strain of the virus. This vaccine did not produce titers in any of the vaccinees.     

Immunogenicity of recombinant fiber-chimeric adenovirus serotype 35 vector-based vaccines in mice and rhesus monkeys

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.     

Impact of prior Dengue immunity on Zika vaccine protection in rhesus macaques and mice

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 ^-/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses.     

Modulation of antigen-specific humoral responses in rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.     

Calcium homeostasis and signaling in the blood-stage malaria parasite

The nature of the mechanisms underlying Ca2+ homeostasis in malaria parasites has puzzled investigators for almost two decades. This review summarizes the current knowledge about Ca2+ homeostasis in Plasmodium spp and highlights some key aspects of this process that are specific to this parasite. Plasmodium spp are exposed, during their intracellular stage, not to the usual millimolar concentrations of Ca2+ found in body fluids, but rather to the very low Ca2+ environment of the host cell cytoplasm. Two crucial questions then arise: (1) how is Ca2+ homeostasis achieved by these protozoa; and (2) do they use Ca2+-based signaling pathways? By critically reviewing the recent literature in the field, Célia Garcia here provides at least some partial answers to these questions.     

Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques

We evaluated three nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of four rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, enzyme-linked immunosorbent assay (ELISA) antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge.     

Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8(+) CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in approximately 1-microm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4(+) and CD8(+) T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30-0.71 and 0.32-2.21% of the circulating CD8(+) and CD4(+) T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.