Time dependence of protective post-exposure prophylaxis with human monoclonal antibodies against pathogenic SHIV challenge in newborn macaques

In a primate model of postnatal virus transmission, we have previously shown that 1 h post-exposure prophylaxis (PEP) with a triple combination of neutralizing monoclonal antibodies (nmAbs) conferred sterilizing protection to neonatal macaques against oral challenge with pathogenic simian-human immunodeficiency virus (SHIV). Here, we show that nmAbs can also partially protect SHIV-exposed newborn macaques against infection or disease, when given as 12 or 24 h PEP, respectively. This work delineates the potential and the limits of passive immunoprophylaxis with nmAbs. Even though 24 h PEP with nmAbs did not provide sterilizing immunity to neonatal monkeys, it contained viremia and protected infants from acute disease. Taken together with our results from other PEP studies, these data show that the success of passive immunization depends on the nmAb potency/dose and the time window between virus exposure and start of immunotherapy.     

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.     

Protective heterologous immunity against fatal ehrlichiosis and lack of protection following homologous challenge

The roles of antibodies and memory T cells in protection against virulent Ehrlichia have not been completely investigated. In this study, we addressed these issues by using murine models of mild and fatal ehrlichiosis caused by related monocytotropic Ehrlichia strains. Mice were primed with either Ehrlichia muris or closely related virulent ehrlichiae transmitted by Ixodes ovatus (IOE) ticks given intraperitoneally or intradermally. All groups were reinfected intraperitoneally, 30 days later, with a lethal high dose of IOE. Priming with E. muris, but not IOE, induced strong CD4+ and CD8+ memory type 1 T-cell responses, Ehrlichia-specific immunoglobulin G (IgG) antibodies, and persistent infection. Compared to IOE-primed mice, subsequent lethal IOE challenge of E. muris-primed mice, resulted in (i) 100% protection against lethal infection, (ii) strong Ehrlichia-specific secondary gamma interferon (IFN-gamma)-producing effector/effector memory CD4+ and CD8+ T-cell responses, (iii) enhanced secondary anti-ehrlichial antibody response, (iv) accelerated bacterial clearance, and (v) the formation of granulomas in the liver and lung. E. muris-primed mice challenged with IOE had lower levels of serum interleukin-1alpha (IL-1alpha), IL-6, and IL-10 compared to unprimed mice challenged with IOE. Interestingly, the fatal secondary response in IOE-primed mice correlated with (i) decline in the Ehrlichia-specific CD4+ and CD8+ type 1 responses, (ii) marked hepatic apoptosis and necrosis, and (iii) substantial bacterial clearance, suggesting that fatal secondary response is due to immune-mediated tissue damage. In conclusion, protection against fatal ehrlichial infection correlates with strong expansion of IFN-gamma-producing CD4+ and CD8+ effector memory type 1 T cells, which appear to be maintained in the presence of IgG antibodies and persistent infection.     

The protective effects of hyperimmune anti-murine cytomegalovirus antiserum against lethal viral challenge: the case for passive-active immunization

The administration of 0.2 ml of hyperimmune anti-mouse cytomegalovirus (CMV) antiserum intraperitoneally (ip) or intravenously provided complete protection against lethal challenge (10(5.8) PFU ip) with murine CMV. Antiserum protection was complete when the antiserum was administered as long as 24 hr after viral challenge. The administration of antiserum had little effect on the titers of virus in the organs of these animals. Ammonium sulfate-treated antiserum provided similar complete protection. Animals rechallenged with 10(6)-10(6.5) PFU of murine CMV 1 month after initial challenge, at a time when the administrated antiserum was no longer detectable, all survived. We conclude that hyperimmune antiserum can provide significant protection against otherwise lethal murine CMV infection, that the protecting material lies within the immunoglobulin fraction, and that long-term immunity results from the combined exposure to virus and antiserum. Such passive-active protection could be useful in protecting against human CMV infection.     

Recombinant GroEL enhances protective antigen-mediated protection against Bacillus anthracis spore challenge

The fatal inhalation infection caused by Bacillus anthracis results from a complex pathogenic cycle involving release of toxins by bacteria that germinate from spores. Currently available vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, these PA-based vaccines are only partially protective against spore challenge in mice. This shows that exclusive elicitation of high anti-PA titer does not directly correlate with protection. Here, we demonstrate that inclusion of GroEL of B. anthracis with PA elicits enhanced protection against anthrax spore challenge in mice. GroEL was included as it has been reported to be present both on the exosporium and in the secretome in addition to the cell surface of B. anthracis. It has also been found protective against other pathogens. In the present study, immunization with GroEL alone was also potent enough to induce high humoral and cell-mediated response and significantly prolonged the mean time to death in spore-challenged mice. As a surface antigen, opsonization of spores with anti-GroEL IgG showed increased uptake of treated spores and therefore accelerated rate of spore destruction by phagocytic cells leading to the protection of mice. We found that GroEL was able to enhance nitric oxide release from lymphocytes and also reduce bacterial load from the organs, probably through the activation of macrophages and over-expression of certain innate immunity receptors. Therefore, the present study emphasizes that GroEL is an effective immunomodulator against B. anthracis infection.     

Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax

Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 mug or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge with B. anthracis in a dose-related manner. The protection conferred by passive transfer of 500 mug macaque IgG correlated significantly (P = 0.003; r = 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 mug of Escherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolized B. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.     

Protective effect of vaginal application of neutralizing and nonneutralizing inhibitory antibodies against vaginal SHIV challenge in macaques

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcγ receptor (FcγR)–mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FcγR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.     

Immunogenic and protective efficacy of recombinant protein GtxA-N against Gallibacterium anatis challenge in chickens

Gallibacterium anatis is a major cause of reproductive tract infections in chickens. Here, we aimed to evaluate the efficacy of the recombinant protein GtxA-N at protecting hens, by addressing three objectives; (i) evaluating the antibody response following immunization (ii) scoring and comparing lesions, following challenge with G. anatis, in immunized and non-immunized hens and (iii) investigating if the anti-GtxA-N antibody titre in individual hens correlated with the observed lesions. Two consecutive experiments were performed in hens. In the first experiment hens were immunized with GtxA-N on day 0 and day 14, infected with G. anatis on day 28 and euthanized on day 56. The GtxA-N antibody response was assessed in pooled serum samples throughout the experiment, using an indirect enzyme-linked immunosorbent assay (ELISA). In the second experiment the GtxA-N antibody titres were assessed in individual hens before and after immunization. Subsequently, the hens were inoculated with G. anatis and finally all hens where euthanized and submitted for post mortem examination 48?h after inoculation. Immunization elicited strong antibody responses that lasted at least 8 weeks (P?G. anatis infection were significantly lower in immunized hens compared to non-immunized hens (P?=?.004). Within the immunized group, no correlation was found between the individual antibody titres and the lesion scores. This study clearly demonstrated GtxA-N as a vaccine antigen able of inducing protective immunity against G. anatis.     

Induction of CD8+ T cell-mediated protective immunity against Trypanosoma cruzi

Trypanosoma cruzi was transformed with the Plasmodium yoelii gene encoding the circum-sporozoite (CS) protein, which contains the well-characterized CD8+ T cell epitope, SYVPSAEQI. In vivo and in vitro assays indicated that cells infected with the transformed T. cruzi could process and present this malaria parasite-derived class I MHC-restricted epitope. Immunization of mice with recombinant influenza and vaccinia viruses expressing the SYVPSAEQI epitope induced a large number of specific CD8+ T cells that strongly suppressed parasitemia and conferred complete protection against the acute T. cruzi lethal infection. CD8+ T cells mediated this immunity as indicated by the unrelenting parasitemia and high mortality observed in immunized mice treated with anti-CD8 antibody. This study demonstrated, for the first time, that vaccination of mice with vectors designed to induce CD8+ T cells is effective against T. cruzi infection.     

Protection against aerosolized Yersinia pestis challenge following homologous and heterologous prime-boost with recombinant plague antigens

A Yersinia pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen against aerosol challenge with Y. pestis in murine studies. In the current study, we examined different prime-boost regimens with F1-V and demonstrate that (i) boosting by a route other than the route used for the priming dose (heterologous boosting) protects mice as well as homologous boosting against aerosol challenge with Y. pestis, (ii) parenteral immunization is not required to protect mice against aerosolized plague challenge, (iii) the route of immunization and choice of adjuvant influence the magnitude of the antibody response as well as the immunoglobulin G1 (IgG1)/IgG2a ratio, and (iv) inclusion of an appropriate adjuvant is critical for nonparenteral immunization.     

Babesia bovis: Vaccination of cattle against heterologous challenge with fractions of lysate from infected erythrocytes

The distilled water lysate of erythrocytes infected withBabesia bovis was separated by gel filtration on Sephadex G200. The void volume fraction or a pool of retained fractions which had immunodiffusion activity were injected into two groups of five cattle. These were challenged 2 weeks after the final vaccination with a heterologous strain ofB. bovis. A control group of five cattle was similarly challenged. Two of the five control animals died from the challenge, whereas none of the vaccinated animals died. There were significant differences in parasitaemia and pathophysiological parameters between the vaccinated groups and the control group.     

Ability of recombinant or native proteins to protect monkeys against heterologous challenge with Plasmodium falciparum.

To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparum malaria, we evaluated recombinant proteins representing sequences from relatively high conserved regions of the precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeys against malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147 to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gp190 and its processed products. All antigens were derived from P. falciparum K1, a Thai isolate, while the challenge strain was Palo Alto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which are almost identical to the K1 sequence, essentially the MAD-20 allelic form of gp190. By 12 days following challenge, each control monkey required drug treatment. Three monkeys injected with p190-3 required therapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapy on day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with native gp190, because of health reasons unrelated to malaria, one was not challenged with parasites and one was removed from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animals ranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and 0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gp190. There was a significant correlation between monkeys which did not require drug treatment and antiparasite antibody. These results may suggest that native gp190 and/or its processed products can provide excellent protection against heterologous challenge and that antibody is important for protection. The challenge for vaccine development is to identify the protective sequence(s).     

Protection of rhesus macaques against lethal Plasmodium knowlesi malaria by a heterologous DNA priming and poxvirus boosting immunization regimen

We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-gamma) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/10(6) PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-gamma responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.     

Intranasal immunization induces long-term protection in mice against a Chlamydia trachomatis genital challenge.

In an attempt to confer long-term protective immunity, BALB/c female mice were immunized intranasally with 10(4) inclusion-forming units (IFU) of the Chlamydia trachomatis mouse pneumonitis biovar (MoPn). Animals were subsequently challenged in the ovarian bursa with 10(5) C. trachomatis MoPn IFU at 60, 120, or 180 days post-intranasal immunization. Two control groups were included in the study. One control was sham immunized and mock challenged, and another group was sham immunized and challenged with 10(5) C. trachomatis MoPn IFU. Vaginal cultures were collected at regular intervals following the intrabursal challenge. In comparison with the sham-immunized mice, the animals that were intranasally immunized with C. trachomatis had significant protection, as shown by a reduction in the number of animals that had positive vaginal cultures and by a decrease in the intensity and length of the shedding. Furthermore, histopathological characterization of the genital tract following challenge, in the three groups of mice, showed a minimal inflammatory infiltrate in the C. trachomatis-immunized animals, when compared with the sham-immunized control group. Subsequently, the three groups of female mice that were challenged at 60, 120 and 180 days postimmunization were mated at 6 weeks following the challenge. Overall, in the mice intranasally immunized with C. trachomatis the fertility rates and the number of embryos were similar to those in the sham-immunized and mock-challenged group. In contrast, there was a significant increase in infertility in the groups of mice that were sham immunized and C. trachomatis challenged. In conclusion, intranasal immunization with C. trachomatis induces long-term protection against a genital challenge as shown by a decrease in the infection and infertility rates when compared with sham-immunized animals. Thus, this model may help to characterize the parameters of the immune response that are important in maintaining long-term protection and may aid in identifying the antigenic determinants involved in eliciting protection.     

Better protective effects in rhesus macaques by combining systemic and mucosal application of a dual component vector vaccine after rectal SHIV89.6P challenge compared to systemic vaccination alone

In this study we investigated the efficacy of a multigenic DNA prime/modified vaccinia Ankara (MVA)boost vaccine approach, followed by mucosal challenge with highly pathogenic simian-human immunodeficiency virus (SHIV) 89.6P, using different routes for vaccine delivery. After three times of DNA priming (SIVmac239, GagPol, and SHIV 89.6P Env) one vaccine group of monkeys was immunized with MVA systemically via intramuscular (IM) and intradermal (ID) application, and in another vaccine group the MVA booster immunization comprised the IM, ID, and atraumatic oral route. Although all vaccinees became infected after intra-rectal challenge with SHIV 89.6P, substantial protection as indicated by lower peak and set point viral loads and unambiguous preservation of CD4 T cells could be achieved. As we could only transiently detect low levels of neutralizing antibodies in some vaccinees, these antibodies did not seem to add to the protection in the vaccinees.Our results indicate that both preventive multigenic DNA prime/MVA booster immunization strategies promote the control of virus replication and protect from disease progression. We also demonstrated that combining mucosal and systemic vaccination mediated better protective effects compared to systemic vaccination alone.     

Role of CD8+ cell-produced anti-viral factors in protective immunity in HIV-2-exposed but seronegative macaques resistant to intrarectal SIVsm challenge

The cell‐mediated immune response is likely to be important in controlling HIV/SIV infection. There is evidence that β‐chemokines and other, as yet unknown, anti‐viral factors play a role in host defence against HIV infection. We reported previously that HIV‐2 exposed but seronegative cynomolgus macaques developed SIV‐specific cytotoxic T lymphocytes and were resistant to mucosal SIV challenge. The aim of this study was to examine CD8⁺ cell‐dependent production of β‐chemokines and other anti‐viral factors in these macaques. The animals, selected from among 17 monkeys enrolled in two separate experiments, were either treated with an anti‐viral drug or immunized passively with HIV‐2 antibody‐positive serum. Three of these monkeys were protected against repeated HIV‐2 challenge and were also able to control SIV infection 3 years later. Control samples were obtained from four macaques that became SIV infected and from 39 naïve animals. The three resistant monkeys showed significantly higher production of RANTES and MIP‐1α than the 39 naïve animals. In addition, SIV infection was suppressed by CD8⁺ cell culture supernatants of these monkeys. However, antibodies to chemokines only partially neutralized CD8⁺ cell‐mediated SIV suppression indicating that the anti‐viral activity observed in these monkeys was the result of combined action of several inhibitory factors.     

Merozoite surface protein 4/5 provides protection against lethal challenge with a heterologous malaria parasite strain

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue of Plasmodium falciparum MSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number of Plasmodium yoelii isolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13 P. yoelii isolates sequenced, even though many of the same isolates showed pronounced variability in their MSP1(19) sequences. Nonsynonymous mutations were detected only for the isolates Plasmodium yoelii nigeriensis N67 and Plasmodium yoelii killicki 193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethal Plasmodium yoelii yoelii YM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein from Plasmodium berghei ANKA and Plasmodium chabaudi adami DS and challenged with P. y. yoelii YM. Interestingly, significant protection was afforded by P. berghei ANKA MSP4/5, which shows 81% sequence identity with P. y. yoelii YM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinant P. c. adami DS MSP4/5, which shows 55.7% sequence identity with P. y. yoelii YM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP1(19) and AMA1.     

Induction of protective immunity in a Syrian hamster model against a cytopathogenic strain of Andes virus

Andes virus (ANDV) is responsible for the Hantavirus Pulmonary Syndrome cases in Argentina and neighboring countries, with moderate to high case-fatality rates. ANDV has some particular features, which make it unique among other members of the Hantavirus genus such as person-to-person transmission and causing a disease similar to Hantavirus Pulmonary Syndrome in the hamster as an animal model. The kinetics of replication in Vero E6 cells of an ANDV strain isolated in Argentina, called Andes/ARG, was studied. Cytopathic effect and the formation of clear plaques were observed and therefore Andes/ARG could be quantified by classic plaque assay. The Andes/ARG strain was found to be highly lethal in Syrian hamsters allowing experiments to demonstrate the protective potential of vaccines. A recombinant nucleocapsid protein of ANDV induced a long lasting antibody response and protective immunity against a homologous challenge, but to a lower extent against heterologous challenge by the Seoul virus.