Protection against filarial infection by 45–49 kDa molecules of Brugia malayi via IFN-γ-mediated iNOS induction

Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators’ release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24–48.64 kDa), F11 (33.44–38.44 kDa) and F12 (28.44–33.44 kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L₃-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1β, IL-10, TGF-β release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1β significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.     

Immunization with inflammatory proteome of Brugia malayi adult worm induces a Th1/Th2-immune response and confers protection against the filarial infection

Mastomys coucha and jirds (Meriones unguiculatus) were immunized with four cytokine-stimulating SDS-PAGE resolved fractions F5 (68–84 kDa), F6 (54–68 kDa), F10 (38–42 kDa) and F14 (20–28 kDa) of Brugia malayi adult worm to determine which of these fractions has the potential to influence the establishment of subsequently introduced B. malayi infection in the animals. The proteins in the fractions were analyzed by 2DE and MALDI-TOF. Immunization with F6 suppressed the establishment of third stage larva (L₃) initiated infection in M. coucha (64%; P < 0.01) and jird (42%; P < 0.01). Survival of intraperitoneally implanted adult worms in M. coucha was lowered by F6 (72%; P < 0.01) and F14 (66%; P < 0.05) but not by F5 and F10. Immunization with F6 intensely upregulated both Th1 (IFN-γ, TNF-α, IL-1β, IL-2, IL-6, IgG1, IgG2a and lymphoproliferation) and Th2 (IgG2b and IL-10) responses and NO release. Immunostimulatory proteins HSP60, intermediate filament protein, and translation elongation factor EF-2 were identified in F6 fraction by 2DE and MALDI. The findings suggest that F6 protects the host from the parasite via Th1/Th2 type responses and thus holds promise for development as a vaccine.     

Disorganized muscle protein-1 (DIM-1) of filarial parasite Brugia malayi: cDNA cloning,expression, purification,structural modeling and its potential as vaccine candidate for human filarial infection

We have recently identified disorganized muscle protein-1 (DIM-1) in one of the proinflammatory fractions of the human filaria Brugia malayi adult worm. The present study was undertaken to characterize B. malayi DIM-1 (DIM-1bm) and explore its vaccine potential. In this study we cloned and expressed the DIM-1bm gene, investigated its sequence homology with other nematodes, constructed in silico structural model, purified the recombinant DIM-1bm (rDIM-1bm) protein, and studied the effect of immunization with rDIM-1bm on the establishment of B. malayi infection in Mastomys coucha. DIM-1bm showed similarity with DIM-1 of Caenorhabditis elegans, Ascaris suum and Loa loa. Structural modeling revealed three immunoglobulin domains in DIM-1bm indicating that it is a member of immunoglobulin superfamily (IgSF) and ‘blastn’ results showed that DIM-1bm coding sequence (CDS) have almost no homology with human and mouse nucleotide sequences. Immunization with rDIM-1bm partially protected M. coucha against establishment of infection as inferred by a low recovery of microfilariae (37–64%) and parasite burden (∼50%). The enhanced activity of macrophages, and IFN-γ and NO responses, and elevated levels of specific IgG, IgG1, IgG2a and IgG2b correlated with parasitological findings. This is the first report on cloning, expression, structural modeling and purification of rDIM-1bm and its ability to partially prevent establishment of B. malayi infection. DIM-1bm's almost complete lack of homology with the human counterpart makes it an attractive protein for exploring its vaccine potential.     

B cell and antibody responses in mice induced by a putative cell surface peptidase of Pneumocystis murina protect against experimental infection

RationalePneumocystis pneumonia is a major cause of morbidity and mortality in HIV-infected subjects, cancer patients undergoing chemotherapy and solid organ transplant recipients. No vaccine is currently available. By chemical labeling coupled with proteomic approach, we have identified a putative surface protein (SPD1, Broad Institute gene accession number PNEG_01848) derived from single suspended P. murina cysts. SPD1 was expressed in an insect cell line and tested for vaccine development.MethodsMice were immunized with SPD1 plus adjuvant MF-59 by subcutaneous injection. Three weeks after the last immunization, CD4+ cells were depleted with anti-CD4 antibody GK1.5. The mice were then challenged with 2 × 10⁵ Pneumocystis organisms. Mice were sacrificed at 4 and 6 weeks after PC challenge. Spleen/lung cells and serum were harvested. B cells and memory B cells were assessed via flow cytometry. Specific Pneumocystis IgG antibody was measured by ELISA before and after challenge. Infection burden was measured as real-time PCR for P. murina rRNA.ResultsNormal mice infected with Pneumocystis mounted a serum IgG antibody response to SPD1. Serum from rhesus macaques exposed to Pneumocystis showed a similar serum IgG response to purified SPD1. SPD1 immunization increased B cell and memory B cell absolute cell counts in CD4-depleted Balb/c mice post Pneumocystis challenge in spleen and lung. Immunization with SPD1 significantly increased specific Pneumocystis IgG antibody production before and after challenge. Mice immunized with SPD1 showed significantly decreased P. murina copy number compared with mice that did not receive SPD1 at 6 weeks after challenge.ConclusionImmunization with SPD1 provides protective efficacy against P. murina infection. SPD1 protection against Pneumocystis challenge is associated with enhanced memory B cell production and higher anti–Pneumocystis IgG antibody production. SPD1 is a potential vaccine candidate to prevent or treat pulmonary infection with Pneumocystis.     

Immunization of pregnant women against pertussis: The effect of timing on antibody avidity

BackgroundThe Centers for Disease Control and Prevention recommend tetanus–diphteria–acellular pertussis (Tdap) immunization during pregnancy, preferably at 27–36 weeks gestation.AimsFirst, to assess the relative avidity index (RAI) of umbilical cord immunoglobulin G (IgG) to pertussis toxin (PT) for newborns of women immunized with Tdap during late pregnancy as compared to unimmunized women. Second, to assess whether there is a preferential period of gestational Tdap immunization that provides the highest RAI of umbilical cord IgG to PT.MethodsRAI of IgG to PT was assessed via an adapted ELISA using NH₄SCN as a dissociating agent.ResultsWe found that newborns of women immunized with Tdap during late pregnancy (n = 52) had higher mean RAI of umbilical cord IgG to PT than those of unimmunized women (n = 8), 73.77% ± 12.08 (95% CI, 70.41–77.13) vs. 50.23% ± 21.32 (95% CI, 32.41–68.06), p < 0.001. Further, the RAI of umbilical cord IgG to PT was significantly higher in newborns of women immunized at 27–30⁺⁶ weeks gestation (n = 20) when compared with newborns of women immunized at 31–36 weeks (n = 22) and >36 weeks (n = 7), 79.53% ± 5.61 (95% CI, 76.91–82.16) vs. 71.56% ± 12.58 (95% CI, 65.98–77.14) vs. 63.93% ± 17.98 (95% CI, 47.31–80.56), p < 0.03.ConclusionGestational Tdap immunization between 27 and 30⁺⁶ weeks resulted in the highest avidity of IgG to PT conveyed at delivery as compared with immunization beyond 31 weeks gestation. Future studies should be conducted to confirm our findings to optimize pertussis-controlling strategies.     

Pathogenesis of Brucella ovis in pregnant mice and protection induced by the candidate vaccine strain B. Ovis ΔabcBA

Ovine brucellosis caused by Brucella ovis is a major cause of reproductive failure in sheep. This study aimed to evaluate transplacental infection and pathogenicity of B. ovis wild type strain ATCC 25,840 (WT B. ovis) and the candidate vaccine strain B. ovis ΔabcBA in pregnant mice. A total of 40 BALB/c mice were equally divided into 4 groups: (i) non immunized and uninfected control mice (3/10 mice became pregnant); (ii) non immunized and challenged with WT B. ovis (5/10 pregnant); (iii) inoculated only with B. ovis ΔabcBA (6/10 pregnant); (iv) immunized with B. ovis ΔabcBA and challenged with WT B. ovis (5/10 pregnant). Female mice bred, and five days after visualization of the vaginal plug, they were inoculated intraperitoneally (ip) with 100 µL of sterile PBS, 100 µL of 1 × 10⁶ CFU of B. ovis ΔabcBA, or 100 µL of 1 × 10⁶ CFU of B. ovis WT, according to each group. At the 17th day of gestation, samples of spleen, liver, uterus, placenta, fetus and mammary gland were obtained for bacteriology, histopathology and immunohistochemistry. Non immunized mice challenged with B. ovis WT developed necrotizing placentitis as well as microgranulomas in the liver and spleen. These findings support the notion that B. ovis infection in pregnant mice induces lesions that are similar to those caused by B. abortus in the same animal model. B. ovis ΔabcBA was not recovered from any of the sampled organs, and it did not cause any gross or microscopic lesions, indicating that it is a safe and attenuated strain in this experimental model. In addition, B. ovis ΔabcBA was induced protective immunity as demonstrated by decreased numbers of B. ovis WT in the liver, uterus and fetuses of immunized mice after the challenge with B. ovis WT.     

Recombinant Lactobacillus casei expressing Clostridium perfringens toxoids α, β2, ε and β1 gives protection against Clostridium perfringens in rabbits

The present study used Lactobacillus casei ATCC 393 as antigen delivery system to express C. perfringens toxoids α-β2-ε-β1 to construct the recombination Lactobacillus casei pPG-2-α-β2-ε-β1/L. casei 393. After being induced by 1% xylose, the specificity and integrity of recombinant strain were determined by Western-blotting. Rabbits as native animal model were immunized orally with pPG-2-α-β2-ε-β1/L. casei 393 and the titers of specific IgG and sIgA were determined by ELISA. The result showed that oral administration with the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in rabbits immunized with pPG-2-α-β2-ε-β1/L. casei 393 was observed. Levels of IL-4 and IFN-γ produced were significantly higher in lymphocytes isolated from the vaccine group than those from the control groups. Flow cytometry assay showed that both the percentages of CD4 + T cells and CD8 + T cells from the vaccine group were significantly increased than the control groups. All these results showed that immunizing with recombinants can elicit both humoral immunity and cellular immunity. Besides, in order to determine the effectiveness of oral immunization with pPG-2-α-β2-ε-β1/L. casei 393, rabbits of vaccine group and control groups were challenged with 1 × LD₁₀₀ unit of culture filtrate of C. perfringens type C and type D toxins respectively. After challenge, 100% of the immunized rabbits survived, while the rabbits of the control group were killed within 48 h. Observation on histopathology showed that histopathological changes were obviously found in heart, liver, spleen, lung, kidney, intestine and brain of rabbits from the control groups, while no apparent histopathological change was observed in the vaccine group. All the results show that pPG-2-α-β2-ε-β1/L. casei 393 can elicit effective immunoprotection against C. perfringens. All of these suggest that the use of pPG-2-α-β2-ε-β1/L. casei 393 can be regarded as candidate for the development of a vaccine against C. perfringens.     

Genetic gradient of a host–parasite pair along a river persisted ten years against physical mobility: Baltic Salmo salar vs. Gyrodactylus salaris

The Atlantic salmon, Salmo salar L., in the Tornio River in the Northern Baltic Sea basin accommodates a monogenean ectoparasite, Gyrodactylus salaris. The aim of the study was to understand the population structure of apparently co-adapted host-parasite system: no parasite-associated mortality has been reported. The parasite burden among salmon juveniles (parr) was monitored along 460 km of the river in 2000–2009. Among the parr, 33.0% were infected (n_fish = 1913). The genetic structure of the parasite population was studied by sequencing an anonymous nuclear DNA marker (ADNAM1, three main genotypes) and mitochondrial CO1 (three clades, six haplotypes). During the ten years, the parasite population was strongly and stably genetically differentiated among up- and downstream nurseries (n_ADNAM1 = 411, F_ST = 0.579; n_CO1 = 443, F_ST = 0.534). Infection prevalence among the smolts migrating to sea was higher than in the sedentary parr populations (82.2%, n_fish = 129). The spatial differentiation observed among the sedentary juveniles was reflected temporally in the smolt run: parasite genotypes dominating the upper part of the river arrived later than downstream dwellers (medians June 4 and June 2) to the trap 7 km from the river mouth. The nuclear and mitochondrial markers were in stable disequilibrium which was not relaxed in the contact zone or among the smolts where the parasite clones often met on individual fish. Only five parasite specimens on smolts (n_worms = 217) were putative recent sexual recombinants. The contribution of extant salmon hatcheries into the infection was negligible. The host salmon population in Tornio River is known to show significant spatial differentiation (F_ST = 0.022). The stable spatial genetic structure of the parasite against the high physical mobility suggested a possibility of local co-adaptation of the host-parasite subpopulations.     

Simultaneous immunization of mice with Omp31 and TF provides protection against Brucella melitensis infection

Brucella vaccines consisting of live attenuated Brucella strains are currently used in livestock, but safety concerns preclude their application in humans. Subunit vaccines have recently emerged as safe and efficacious alternatives in both humans and animals. In this study, subunit vaccines were developed that consisted of a recombinant outer membrane protein (rOmp31) and the trigger factor chaperone protein (rTF) of Brucella melitensis, either alone or in combination. BALB/c mice that were immunized with rOmp31 + rTF showed comparable but slightly higher TF-specific IgG1 and IgG2a antibodies as compared to mice with rTF alone. Indeed, mice given this combination had titers of rOmp31-specific antibodies similar to those immunized with rOmp31 alone. In lymphocyte reactivation experiments, the splenocytes of immunized mice, whether given either of these antigens alone or as a cocktail, exhibited a strong antigen-specific recall proliferative response and expressed high amounts of IFN-γ, IL-12, IL-10 and IL-6. Both rTF and rTF + rOmp31 vaccinated mice exhibited significantly higher CD4 and CD8 levels compared to the PBS group. The combination of rOmp31 and rTF provided protection against B. melitensis infection comparable to that of vaccine strain Rev.1. In comparison to rTF alone, combination of rTF and rOmp31 caused only a slight increase in protection level. Although combination of rTF and rOmp31 caused a non-significant increase in IFN-γ induction, antibody level, proliferation index and CD4 and CD8 frequencies compared to rTF alone, its cumulative effects on aforesaid parameters may be viewed as a better efficacy.     

Specific memory B cell response and participation of CD4+ central and effector memory T cells in mice immunized with liposome encapsulated recombinant NE protein based Hepatitis E vaccine candidate

BackgroundLiposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys.MethodsThe current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420 days post immunization (PI) with 5 μg rNEp encapsulated in liposome based adjuvant (2 doses, 4 weeks apart). Mice immunized with a lower dose (1 μg) were assessed only for anamnestic response post booster dose.ResultsVaccine candidate immunized mice (5 μg dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1 μg dose. A booster dose yielded a heightened antibody response in mice with both high (>800 GMT, 5 μg) and low (⩽100 GMT, 1 μg) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4⁺ central and effector memory T cells were high in mice with high anti-HEV IgG titers only.ConclusionsTaken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4⁺ memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human.     

Immunogenicity and therapeutic effects of recombinant Ag85AB fusion protein vaccines in mice infected with Mycobacterium tuberculosis

The immune function of tuberculosis (TB) patients is disordered. By using immune regulators to assist chemotherapy for TB the curative effect might be improved. In this study, a vaccine containing Mycobacterium tuberculosis (M. tuberculosis) recombinant Ag85AB fusion protein (rAg85AB) was constructed and evaluated. The mice were immunized intramuscularly three times at two-week intervals with Ag85AB fusion protein combined with Corynebacterium parvum adjuvant (rAg85AB+CP). In comparison to control mice that received either CP alone or saline, the mice that received rAg85AB+CP had significantly higher number of T cells secreting IFN-γ and higher levels of specific antibodies of IgG, IgG1 and IgG2a isotypes in sera. The specific antibodies also had higher ratios of IgG2a to IgG1, indicating a predominant Th1 immune response. To test for immunotherapy of TB, M. tuberculosis infected mice were given three intramuscular doses of 20 μg, 40 μg or 60 μg of rAg85AB in rAg85AB+CP, or phosphate-buffered saline (PBS), or CP or Mycobacterium phlei (M. Phlei) F.U.36. Compared with the PBS group, 20 µg, 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups reduced the pulmonary bacterial loads by 0.13, 0.15, 0.42 and 0.40 log₁₀, and the liver bacterial loads by 0.64, 0.64, 0.53 and 0.61 log₁₀, respectively. Pathological changes of lungs were less, and the lesions were limited to a certain extent in 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups. These results showed that rAg85AB+CP had immunotherapeutic effect on TB, significantly increasing the cellular immune response, and inhibiting the growth of M. tuberculosis.     

Short-term toxicity tests on the harpacticoid copepod Tisbe battagliai: Lethal and reproductive endpoints

Short-term bioassays based on lethal and reproductive responses of Tisbe battagliai were applied to determine responses of copepods to copper and LAS. Percentage of spawning females, fecundity (F), and total newborn production (N) for 48 and 72 h were calculated for both substances. It was observed percentage of spawning females was not affected by sublethal concentrations of both compounds. Following values were obtained: EC₅₀(N)-48 h of 670±30 μg LAS L^?1 and EC₅₀(F)-48 h of 670±30 μg LAS L^?1; and EC₅₀(N)-72 h of 44.5±1.8 μg Cu L^?1 and EC₅₀(F)-72 h of 30.8±1.1 μg Cu L^?1. Lethal effects of the two substance-types were also assessed, obtaining the LC₅₀-24 h of 1980±160 μg LAS L^?1; and LC₅₀-48 h of 83.1±10.5 μg Cu L^?1 for nauplii; and LC₅₀-72 h of 157±25 μg Cu L^?1, and LC₅₀-72 h of 2660±270 μg LAS L^?1 for adults. Fecundity and total newborn production are sensitive endpoints for determining effects of toxicants.     

Nasal immunization of mice with AFCo1 or AFPL1 plus capsular polysaccharide Vi from Salmonella typhi induces cellular response and memory B and T cell responses

The response to infection against Salmonella involves both B and T cell mediated immunity. An effective immunization can activate an adequate immune response capable to control the primary infection and protect against a secondary infection. Mucosal vaccination, by inducing local pathogen-specific immune responses, has the potential to counter mucosally transmitted pathogens at the portal of entry, thereby increasing the efficacy of vaccines. The aim of this work was to explore the efficacy of AFCo1 or AFPL1, as mucosal adjuvants to stimulate cell immunity and memory responses against Vi polysaccharide antigen of Salmonella typhi (PsVi). Mice immunized with 3 intranasal doses exhibited high levels of PsVi-specific IgG (p < 0.05), IgG2a and IgG2c subclasses. Also, an amplified recall response after a booster immunization with a plain polysaccharide vaccine was induced. Avidities index were higher in mice immunized with adjuvanted formulations at different chaotropic concentrations. Furthermore, IL-12 and IFN-γ levels in nasally vaccinated mice with both adjuvants were induced. Moreover, priming with 3 doses followed by booster immunization with VaxTyVi^® resulted in high levels of anti-Vi specific IgG, IgG subclasses and antibody avidity. Long lived plasma cells in bone marrow, memory B cells and long-term memory T cells after booster dose were induced. The combined formulation of Vi polysaccharide with mucosal adjuvants provides an improved immunogenicity, in particular with regard to cellular responses and long lasting cells responses.     

Recombinant L7/L12 protein entrapping PLGA (poly lactide-co-glycolide) micro particles protect BALB/c mice against the virulent B. abortus 544 infection

Brucella abortus is the etiologic agent of Brucellosis, a zoonotic infection affecting a wide range of animals. It is a highly infectious disease of pandemic potential reporting over 500,000 new human cases annually. Till date, there is no reported vaccine for humans and the available animal vaccines are unsafe, therefore a safe and effective subunit vaccine is highly sought for. In this study, we have evaluated rL7/L12 protein encapsulated in microparticles of PLGA (85:15), a biocompatible and biodegradable polymer approved by FDA for human use. In this work, BALB/c mice have been immunized with rL7/L12 entrapped in microparticles in a prime-boost regimen. Further, evaluation of the immunogenicity of the formulation showed that the IgG antibody titre reached a maxima of 2.2 × 10⁵ (p value 0.0001 v/s control) after the injection of the booster dose. A mixed IgG isotype profile (IgG1/IgG2a) indicated the stimulation of both the cellular as well as humoral immunity which has increased parallely and gradually since the first immunization. High levels of IFN-γ, 815 ± 55 pg/ml were recorded depicting an optimal elicitation of the cellular wing of immunity leading to clearance of splenic bacteria upto 1.69 log units.     

Prime-booster vaccination of cattle with an influenza viral vector Brucella abortus vaccine induces a long-term protective immune response against Brucella abortus infection

This study analyzed the duration of the antigen-specific humoral and T-cell immune responses and protectiveness of a recently-developed influenza viral vector Brucella abortus (Flu-BA) vaccine expressing Brucella proteins Omp16 and L7/L12 and containing the adjuvant Montadine Gel01 in cattle. At 1 month post-booster vaccination (BV), both humoral (up to 3 months post-BV; GMT IgG ELISA titer 214 ± 55 to 857 ± 136, with a prevalence of IgG2a over IgG1 isotype antibodies) and T-cell immune responses were observed in vaccinated heifers (n = 35) compared to control animals (n = 35, injected with adjuvant/PBS only). A pronounced T-cell immune response was induced and maintained for 12 months post-BV, as indicated by the lymphocyte stimulation index (2.7 ± 0.4 to 10.1 ± 0.9 cpm) and production of IFN-γ (13.7 ± 1.7 to 40.0 ± 3.0 ng/ml) at 3, 6, 9, and 12 months post-BV. Prime-boost vaccination provided significant protection against B. abortus infection at 3, 6, 9 and 12 months (study duration) post-BV (7 heifers per time point; alpha = 0.03–0.01 vs. control group). Between 57.1 and 71.4% of vaccinated animals showed no signs of B. abortus infection (or Brucella isolation) at 3, 6, 9 and 12 months post-BV; the severity of infection, as indicated by the index of infection (P = 0.0003 to <0.0001) and rates of Brucella colonization (P = 0.03 to <0.0001), was significantly lower for vaccinated diseased animals than appropriate control animals. Good protection from B. abortus infection was also observed among pregnant vaccinated heifers (alpha = 0.03), as well as their fetuses and calves (alpha = 0.01), for 12 months post-BV. Additionally, 71.4% of vaccinated heifers calved successfully whereas all pregnant control animals aborted (alpha = 0.01). Prime-boost vaccination of cattle with Flu-BA induces an antigen-specific humoral and pronounced T cell immune response and most importantly provides good protectiveness, even in pregnant heifers, for at least 12 months post-BV.     

Mucosal immunization confers long-term protection against intragastrically established Neospora caninum infection

Neospora caninum is an obligate intracellular protozoan parasite responsible for heavy economic losses in dairy and beef cattle farms worldwide. Although vaccination is widely regarded as the preferable strategy to prevent neosporosis no commercial vaccine is currently available. We have previously shown that intranasal immunization with an N. caninum antigen extract enriched in hydrophobic proteins plus CpG adjuvant protected mice against intragastrically established neosporosis. Nevertheless, the antigen specificity as well as the long-term protective effect of this immunization strategy were not determined. Here, we show that the protective effect of this intranasal immunization procedure lasted for at least 20 weeks. Protection was accompanied by long-lasting elevated levels of parasite-specific serum IgG and intestinal IgA. Moreover, spleen and mesenteric lymph node cells obtained from non-infected long-term immunized mice responded by producing interferon-γ following in vitro parasite-antigen recall. Analysis of serum IgG and intestinal IgA antibody reactivity in immunized mice identified dense granule antigen 7 (NcGRA7) and microneme associated protein 1 (NcMIC1) as immunodominant antigens respectively recognized by those antibody fractions. In summary, this work shows that a previously reported mucosal immunization strategy against N. caninum infection established through the gastrointestinal tract is effective in the long term.     

Hepatitis B vaccination uptake and correlates of serologic response among HIV-infected and uninfected men who have sex with men (MSM) in Bangkok,Thailand

BackgroundVaccination against hepatitis B virus (HBV) is recommended for all HBV-susceptible men who have sex with men (MSM). There is limited information on correlates of immunity to HBV vaccination in this group. We present serologic response rates to hepatitis B vaccine and identify factors associated with impaired response among HIV-uninfected and HIV-infected Thai MSM.MethodologyHBV-susceptible volunteers were offered hepatitis B vaccination at months zero, one, and six. We measured baseline (pre-vaccination) total serum IgG and IgG subclasses (all participants), baseline CD4 count, and plasma HIV-1 viral load (PVL) (HIV+ participants). HBV serologies were retested at 12 months. Serologic responses were compared between all groups in men receiving three vaccine doses.Results511/651 HIV-negative and 64/84 HIV-positive participants completed the three-dose series. Response rates in HIV-uninfected and -infected participants were 90.1% vs. 50.0% (p < 0.0001). Median pre-vaccination IgG was higher among non-responders than responders overall (1238.9.0 vs. 1057.0 mg/dL, p = 0.003) and among HIV-infected participants (1534.0 vs. 1244.5 mg/dL, p = 0.005), but not significantly among HIV-uninfected participants (1105.5 vs. 1054.3 mg/dL, p = 0.96). Pre-vaccination IgG1 and IgG3 levels were higher among HIV-positive than HIV-negative participants (median 866.0 vs. 520.3, and 105.8 vs. 83.1 mg/dL, respectively, p < 0.0001). Among HIV-infected participants, median CD4 count in non-responders was 378 cells/μL vs. 431 cells/μL in responders (p = 0.20). Median PVL in non-responders was 64,800 copies/mL vs. 15500 copies/mL in responders (p = 0.04). Participants with pre-vaccination plasma IgG >1550 mg/dL and PVL >10,000 copies/mL were almost always non-responsive (p < 0.01).ConclusionsHIV infection was associated with poor vaccine responses. High plasma viral load, elevated pre-vaccination total serum IgG and elevated pre-vaccination IgG1 are associated with poorer response to vaccination among HIV-infected MSM. In this group, the combination of high PVL and pre-vaccination total IgG is highly predictive of vaccine failure.     

Enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant,rOv-ASP-1

Immunization is the best way to prevent seasonal epidemics and pandemics of influenza. There are two kinds of influenza vaccines available in the United States: an inactivated vaccine (TIV) and an attenuated vaccine; however, only TIV is approved for immunization of the elderly population. While the aged population has the highest rate of influenza vaccination, the protective efficacy is low as evidenced by elderly individuals having the highest mortality associated with influenza. Recently, we reported that an adjuvant derived from the helminth parasite Onchocerca volvulus, named O. volvulus activation-associated secreted protein-1 (Ov-ASP-1), can significantly enhance the protective efficacy of an inactivated vaccine (TIV) in young adult mice. In the current study, we examined whether this recombinant Ov-ASP-1 (rOv-ASP-1) can enhance the efficacy of TIV in aged mice as well. While primary immunization with TIV alone produced only a low level of influenza-specific antibodies (total IgG, IgG1, and IgG2c) in aged mice, the antibody levels were significantly increased after immunization with TIV + rOv-ASP-1. More importantly, the level of the total IgG in aged mice administered TIV + rOv-ASP-1 was comparable to that of young adult mice immunized with TIV alone. Co-administration of rOv-ASP-1 induced a low level of cross-reactive antibody and enhanced the protective efficacy of TIV in aged mice, reflected by significantly increased survival after challenge with a heterologous influenza virus. rOv-ASP-1 was also superior to the conventional adjuvant alum in inducing specific IgG after TIV immunization in aged mice, and in conferring protection after challenge. These results demonstrate that rOv-ASP-1 may serve as a potential adjuvant for influenza vaccine to improve the efficacy of protection in the elderly.     

Recombinant Ax21 protein is a promising subunit vaccine candidate against Stenotrophomonas maltophilia in a murine infection model

Stenotrophomonas maltophilia is an emerging pathogen that can cause several disease manifestations such as bacteremia, meningitis, respiratory tract infections and others. More seriously, this pathogen has a highly evolving antibiotic resistance profile. Antibiotic misuse is further aggravating the situation by inducing the development of multi- and even pan-resistance. Thus, employing diverse strategies to overcome this increasing antibiotic resistance is of paramount importance. In general, vaccination is one of these strategies that prevents the onset of infection, provides long term protection against infection, and most importantly diminishes the antibiotic consumption, thus, resulting in controlling resistance. Unfortunately, vaccine research concerning S. maltophilia is very scarce in the literature. Ax21 protein is an outer membrane protein implicated in several virulence mechanisms of S. maltophilia such as quorum sensing, biofilm formation, and antibiotic resistance. Our computational analysis of Ax21 revealed its potential immunogenicity. In the current study, Ax21 protein of S. maltophilia was cloned and heterologously expressed in Escherichia coli. Mice were immunized with the purified recombinant antigen using Bacillus Calmette-Guérin (BCG) and incomplete Freund’s adjuvant (IFA) as immune-adjuvants. Enzyme-linked immunosorbent assay (ELISA) revealed significant antigen-specific IgG1, IgG2a and total IgG levels in immunized mice which reflected successful immune stimulation. Immunized mice that were challenged with S. maltophilia showed a substantial reduction in bacterial bioburden in lungs, liver, kidneys, and heart. In addition, liver histological examination demonstrated a remarkable decrease in pathological signs such as necrosis, vacuolation, bile duct fibrosis and necrosis, infiltration of inflammatory cells, and hemorrhage. Whole cell ELISA and opsonophagocytic assay confirmed the ability of serum antibodies from immunized mice to bind and facilitate phagocytosis of S. maltophilia, respectively. To our knowledge, this is the first report to demonstrate the vaccine protective efficacy of Ax21 outer membrane protein against S. maltophilia infection.