The molecular characterization and immune protection of adhesion protein 65 (AP65) of Trichomonas vaginalis.

BACKGROUND: Adherence to the surface of the host cell is the precondition for T. vaginalis parasitism and pathogenicity, causing urogenital infection. The AP65 of T. vaginalis (TvAP65) involves in the process of adhesion. So, the present study was aimed at investigating the molecular characterization and vaccine candidacy of TvAP65 for protecting the host from the onset of Trichomoniasis. METHODS: The open reading frame (ORF) of TvAP65 was amplified and then inserted into pET-32a (+) to clone recombinant TvAP65 (rTvAP65). The immunoblotting determined the immunogenicity and molecular size of TvAP65, while immunofluorescence staining visualized and the precise localization of TvAP65 in T. vaginalis trophozoites. Animal challenge and enzyme-linked immunosorbent assay (ELISA) test were used to evaluate the immunoprotection and the types of the immune response of TvAP65. RESULTS: By the sequence analysis, TvAP65 encoded a 63.13 kDa protein that consisted 567 amino acid residues with a high antigenic index. The western blotting revealed that rTvAP65 and native TvAP65 could interact with the antibodies in the rat serums post hoc rTvAP65 immunization and the serums from the mice that were experimentally infected with T. vaginalis, respectively. Immunofluorescence stained TvAP65 on the surface of T. vaginalis trophozoites. Moreover, following emulsification with Freund's adjuvant, rTvAP65 was subsequently administered to BALB/c mice three times at 0, 2, and 4 weeks and the results from this animal challenge experiments showed significant increases in immunoglobulins of IgG2a, IgG1, and IgG, and cytokine of IFN-γ, and IL-2, and 10. Lastly, rTvAP65 vaccinated animals had a prolonged survival time (26.80 ± 4.05) after challenged by T. vaginalis. CONCLUSIONS: TvAP65 mediated the adhesion of T. vaginalis to the host epithelia for the pathogenesis of the parasite and can be considered as a candidate protein for designing a functional vaccine that induces cell-mediated and humoral immunity against the T. vaginalis infection.

The Molecular Characterization and Immunity Identification of Rhoptry Protein 22 of Toxoplasma gondii as a DNA Vaccine Candidate Against Toxoplasmosis.

Toxoplasma gondii (T. gondii) rhoptry proteins (TgROPs) have been considered main targets and indicator molecules for immune diagnosis and prophylaxis since they initially present during the process of invasion. In this study, the effect of intramuscularly injecting the genetic vaccine pVAX-ROP22 was evaluated, made by inserting the TgROP22 sequence into the eukaryotic expression vector of pVAX I, into BALB/c mice. The levels of IgG, IgG1, and IgG2a in pVAX-ROP22 vaccinated animals were integrally increased. It was uncovered by cytokine profile analyses that the levels of IFN-γ and IL-2 were significantly increased, while no significant changes were detected in IL-4 and IL-10 levels. In addition, we found that immunization with pVAX-ROP22 significantly prolonged the survival time (13.80 ± 1.75 d) of mice after challenge infection with the virulent T. gondii RH strain, in comparison with those of control animals (died within 10 d). Moreover, the number of brain cysts (1,406 ± 277) in the animals subjected to pVAX-TgROP22 vaccination decreased remarkably (P < 0.05) compared with the blank control mice (2,333 ± 473), and the size of brain cysts in pVAX-TgROP22 group was significantly smaller than the groups of blank, PBS and pVAXI. These results suggested that TgROP22 as DNA vaccine could trigger strong humoral and cellular responses and induce partial protection against toxoplasmosis.

Establishment of an animal challenge model as a potency assay for an inactivated Enterovirus Type 71 vaccine.

Enterovirus 71 (EV71) belongs to the Enterovirus genus of the Picornaviridae family, and its occurrence in Asia is associated with hand-foot-and-mouth disease (HFMD), leading to death in some cases, in young children. An effective EV71 vaccine is therefore urgently needed. In this study, we established a two-step EV71 vaccine potency model. Intraperitoneal injections in 2-day-old suckling mice were used to establish the LD50 of EV71 B4, B5, C2, C4, and C5 subgenotypes. Only C4 caused hind limb paralysis in mice (LD50: 2.62 ± 0.45). EV71 VP1 protein was identified in the brain tissues at histology. In the second phase of the model, 3-week-old female ICR mice received one primary and two boosting i.p. injections of formalin-inactivated EV71 B4 and C4 vaccine. Immunized serum was neutralized in vitro with EV71 C4 and applied to the murine challenge model. The C4 vaccine-immunized serum exhibited the highest protective titre (ED50 = 114.6), while the B4 immunized serum had the weakest protective titre (ED50 = 34.3). Additionally, human plasma and intravenous immunoglobulin displayed significant protection in the neutralization assay. Our results could facilitate candidate EV71 vaccine immunogenicity and efficacy evaluations, and may help establish reference EV71 antisera in the future.

Beta RBD boost broadens antibody-mediated protection against SARS-CoV-2 variants in animal models.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with resistance to neutralizing antibodies are threatening to undermine vaccine efficacy. Vaccination and infection have led to widespread humoral immunity against the pandemic founder (Wu-Hu-1). Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet clear whether heterotypic boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. We show, in macaques immunized with Wu-Hu-1 spike, that a single dose of adjuvanted beta variant receptor binding domain (RBD) protein broadens neutralizing antibody responses to heterologous VOCs. Passive transfer of plasma sampled after Wu-Hu-1 spike immunization only partially protects K18-hACE2 mice from lethal challenge with a beta variant isolate, whereas plasma sampled following heterotypic RBD boost protects completely against disease.

Identification of Toxoplasma Gondii Tyrosine Hydroxylase (TH) Activity and Molecular Immunoprotection against Toxoplasmosis.

The neurotropic parasite Toxoplasma gondii (T. gondii) infection can change the behavior of rodents and cause neuropsychological symptoms in humans, which may be related to the change in neurotransmitter dopamine in the host brain caused by T. gondii infection. T. gondii tyrosine hydroxylase (TgTH) is an important factor in increasing the neurotransmitter dopamine in the host brain. In this study, the enzyme activity of TgTH catalytic substrate for dopamine production and the molecular characteristics of TgTH were identified. In order to amplify the open reading frame (ORF), the designing of the specific primers for polymerase chain reaction (PCR) was on the basis of the TgTH sequence (GenBank Accession No. EU481510.1), which was inserted into pET-32a (+) for the expression of recombined TgTH (rTgTH). The sequence analysis indicated that the gene of TgTH directed the encoding of a 62.4-kDa protein consisting of 565 amino acid residues, which was predicted to have a high antigen index. The enzyme activity test showed that rTgTH and the soluble proteins extracted separately from T. gondii RH strain and PRU strain could catalyze the substrate to produce dopamine in a dose-dependent manner, and the optimum catalytic temperature was 37 °C. The result of the Western Blotting assay revealed that the rTgTH and the native TgTH extracted from somatic of T. gondii RH tachyzoite were successfully detected by the sera of mice infected with T. gondii and the rat serum after rTgTH immune, respectively. Immunofluorescence analysis using antibody against rTgTH demonstrated that the protein was expressed and located on the surface of T. gondii RH tachyzoite. Freund's adjuvant was used to emulsify the rTgTH, which was subsequently applied to BALB/c mouse immune thrice on week 0, week 2, and week 4, respectively. The result of the animal challenge experiments showed an integral increase in IgG, IgG2a, IgG1, and IFN-γ, IL-4, and IL17 were as well significantly increased, and that the rTgTH vaccinated animals apparently had a prolonged survival time (14.30 ± 2.41) after infection with the RH strain of T. gondii compared with that of the non-vaccinated control animals, which died within 11 days. Additionally, in the rTgTH vaccination group, the number of brain cysts (1275 ± 224) significantly decreased (p < 0.05) compared to the blank control group (2375 ± 883), and the size of the brain cysts in the animals immunized with rTgTH vaccination was remarkably smaller than that of the control mice. All the findings prove that TgTH played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity.

The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33).

Trichomoniasis is caused by Trichomonas vaginalis (T. vaginalis), which is a widespread and serious sexually transmitted pathogen in humans. The procedure of T. vaginalis adherence to the host cell is the precondition for T. vaginalis parasitism and pathogenicity. The AP33 adhesin of T. vaginalis (TvAP33) plays a key role in the process of adhesion. In this study, the specific primers for polymerase chain reaction (PCR) were designed based on the sequence of TvAP33 (GenBank Accession No. U87098.1) to amplify the open reading frame (ORF), and the ORF was inserted into pET-32a (+) to produce recombinant TvAP33 (rTvAP33). The sequence analysis indicated that the TvAP33 gene encoded a protein of 309 amino acids with 32.53 kDa, and the protein was predicted to have a high antigen index. Western blotting assay showed rTvAP33 was successfully recognized by the sera of mice experimentally infected with T. vaginalis, while native TvAP33 in the somatic extract of T. vaginalis trophozoite was as well detected by sera from rats immunized with the rTvAP33. Immunofluorescence analysis using an antibody against rTvAP33 demonstrated that the protein was expressed and located on the surface of T. vaginalis trophozoites. The recombinant protein was emulsified in Freund's adjuvant and used to immunize BALB/C mice three times at days 0, 14, and 28. The result of animal challenge experiments revealed the levels of IgG, IgG1, and IgG2a, and IL-4, IL-10, and IL17 among rTvAP33 vaccinated animals were integrally increased. Moreover, the rTvAP33 vaccinated animals were apparently prolonged survival time (26.45 ± 4.10) after challenge infection with this parasite. All these results indicated that TvAP33 could be used as vaccine candidate antigen to induce cell-mediated and humoral immunity.

Porcine reproductive and respiratory syndrome virus NADC30-like strain accelerates Streptococcus suis serotype 2 infection in vivo and in vitro.

Porcine reproductive and respiratory syndrome (PRRS), an economically significant pandemic disease, commonly results in increased impact of bacterial infections, including those by Streptococcus suis (S. suis). In recent years, PRRS virus (PRRSV) NADC30-like strain has emerged in different regions of China, and coinfected with S. suis and PRRSV has also gradually increased in clinical performance. However, the mechanisms involved in host innate responses towards S. suis and their implications of coinfection with NADC30-like strain remain unknown. Therefore, the pathogenicity of NADC30-like strain and S. suis serotype 2 (SS2) coinfection in vivo and in vitro was investigated in this study. The results showed that NADC30-like increased the invasion and proliferation of SS2 in blood and tissues, resulting in more severe pneumonia, myocarditis, and peritonitisas well as higher mortality rate in pigs. In vitro, NADC30-like strain increased the invasion and survival of SS2 in porcine alveolar macrophages (PAM) cells, causing more drastic expression of inflammatory cytokines and activation of NF-ĸB signalling. These results pave the way for understanding the interaction of S. suis with the swine immune system and their modulation in a viral coinfection.

Immune Protection of Rhoptry Protein 21 (ROP21) of Toxoplasma gondii as a DNA Vaccine Against Toxoplasmosis.

Toxoplasma gondii rhoptry proteins (TgROPs) are the major targets as key molecules for immunodiagnosis as well as immunoprophylaxis because of their initial presentation to the host immune system. In this work, it was aimed at evaluating the protection effect of TgROP21 DNA vaccine on experimental mice subjected to T. gondii challenge. The gene sequence encoding TgROP21 was inserted into the eukaryotic expression vector pVAX I, and western blotting indicates that the lysate of BHK cells transfected with pVAX-TgROP21 was specifically recognized as a band of about 82.6 kDa by serum obtained from a T. gondii infected chicken. The efficacy of intramuscular vaccination of BALB/c mice three times at weeks 0, 2, and 4 with pVAX-ROP21 was analyzed. The levels of IgG, IgG1, and IgG2a among pVAX-ROP21 vaccinated animals were integrally increased. It was uncovered by cytokine profile analyses that IFN-γ was significantly increased, while no significant changes were detected in interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10). Additionally, we found that immunization with pVAX-ROP21 significantly prolonged survival time (13.50 ± 1.65 days) after challenge infection with the virulent T. gondii RH strain, in comparison to those of control animals (died within 10 days). Moreover, the number of brain cysts (1475 ± 163) in the animals subjected to pVAX-TgROP21 vaccination decreased remarkably (P < 0.05) compared to the blank control mice (2333 ± 473), and the size of brain cysts in pVAX-TgROP21 group was significantly smaller than the groups of blank, PBS and pVAXI. It was indicated that intense cell-mediated and humoral immunity was triggered and defense against T. gondii was partially induced after vaccination by TgROP21.

Molecular characterization and protective immunity of rhoptry protein 35 (ROP35) of Toxoplasma gondii as a DNA vaccine.

Toxoplasma gondii rhoptry proteins (TgROPs) have been considered the main targets and indicator molecules for immune diagnosis and prophylaxis because they present during the initial process of invasion. The aim of this study was to evaluate the protective effect of a TgROP35 DNA vaccine on experimental mice subjected to T. gondii challenge. The effect of intramuscularly injecting the genetic vaccine pVAX-ROP35 into BALB/c mice was evaluated, by first inserting the TgROP35 sequence into the pVAX I eukaryotic expression vector. The levels of IgG, IgG1 and IgG2a in pVAX-ROP35-vaccinated animals were integrally increased. Cytokine profile analyses revealed that IFN-γ, IL-2 and IL-10 levels were significantly increased, while there were no significant differences in IL-4 expression between the pVAX-ROP35 immunized and control groups. Additionally, we found that immunization with pVAX-ROP35 significantly prolonged the survival time (13.90 ±â€¯1.97 days) of mice after challenge infection with the virulent T. gondii RH strain compared with that in non-vaccinated control animals (mice died within 10 days). Moreover, the number of brain cysts (1450 ±â€¯287) in the animals subjected to pVAX-TgROP35 vaccination decreased remarkably (P <  0.05) compared to that in the blank control mice (2333 ± 473). Furthermore, the size of brain cysts in the pVAX-TgROP35 group was significantly smaller than that in the blank control, PBS and pVAXI groups. The findings indicated that intense cell-mediated and humoural immunity was triggered and that defence mechanisms against T. gondii were partially induced after vaccination by TgROP35.

Evaluation of Recombinant Multi-Epitope Outer Membrane Protein-Based Klebsiella pneumoniae Subunit Vaccine in Mouse Model.

Safety and protective efficacy of recombinant multi-epitope subunit vaccine (r-AK36) was evaluated in a mouse model. Recombinant AK36 protein comprised of immunodominant antigens from outer membrane proteins (Omp's) of Klebsiella pneumoniae namely OmpA and OmpK36. r-AK36 was highly immunogenic and the hyperimmune sera reacted strongly with native OmpA and OmpK36 proteins from different K. pneumoniae strains. Hyperimmune sera showed cross-reactivity with Omp's of other Gram-negative organisms. Humoral responses showed a Th2-type polarized immune response with IgG1 being the predominant antibody isotype. Anti-r-AK36 antibodies showed antimicrobial effect during in vitro testing with MIC values in the range of 25-50 µg/ml on different K. pneumoniae strains. The recombinant antigen elicited three fold higher proliferation of splenocytes from immunized mice compared to those with sham-immunized mice. Anti-r-AK36 antibodies also exhibited in vitro biofilm inhibition property. Subunit vaccine r-AK36 immunization promoted induction of protective cytokines IL-2 and IFN-γ in immunized mice. When r-AK36-immunized mice were challenged with 3 × LD100 dose, ∼80% of mice survived beyond the observation period. Passive antibody administration to naive mice protected them (67%) against the lethal challenge. Since the targeted OMPs are conserved among all K. pneumoniae serovars and due to the strong nature of immune responses, r-AK36 subunit vaccine could be a cost effective candidate against klebsiellosis.