Oral Immunization for Anti-snake Venom Production in Equine Animals

The immunization process of current commercial manufacturing of anti-snake venom (ASV), uses injections of bentonite, complete Freund’s adjuvant, or incomplete Freund’s adjuvant, mixed with low doses of the snake venom in horses (but rarely in other large mammals), which frequently cause serious adverse effects in host animals. At the site of injection, horses may develop painful swelling, granuloma, abscess, scar, or systemic neurological and hematological defects, low antibody response, or death due to anaphylactic shock. We sought to investigate a novel alternate immunization strategy with oral administration of snake venom with adjuvants. We utilized M5904 mineral oil emulsion as an adjuvant that was mixed with sub-lethal doses (LD) of the snake venoms. Our preliminary experiments were initiated in March 2011 and the present data culminated in March 2018. In our initial experiments which were carried out in inbred mice, the LD100 was 10.36 ug/25 grams of mice for Naja. oxins and 10.0 ug/25 gram of Naja. karachians. We extrapolated the sub-LD dose to horses by cutting the LD100 in mice to 20%. This dose did not cause any apparent pathology in horses and therefore, we adopted that dose for the equine.

Oral Immunization of FMDV Vaccine Using pH-Sensitive and Mucoadhesive Thiolated Cellulose Acetate Phthalate Microparticles

Several barriers such as gastric pH, enzymatic degradation and rapid transit should be overcome to orally deliver antigens for taking up by epithelial microfold cells in Peyer's patches of small intestine. To solve the above mentioned problems, we designed pH-sensitive and mucoadhesive polymeric microparticles (MPs) prepared by double emulsion technique using cellulose acetate phthalate (CAP) to enhance immune response of foot-and-mouth disease (FMD) virus (FMDV) subunit vaccine. Thiolation of CAP improved mucoadhesive property of CAP to prolong the MPs transit time through the gastrointestinal tract. Thiolated CAP (T-CAP) also slowed down antigen release in acidic pH of stomach but released more antigens in neutral pH of small intestine due to the pH-sensitivity of the T-CAP. Oral immunization of a chimerical multi-epitope recombinant protein as the FMD subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer's patches eliciting mucosal IgA response. It will make a step forward into a promising oral subunit vaccine development in livestock industry.

Eosinophils are Required for Immune Responses Induced by Oral Immunization

Eosinophils are multifunctional leukocytes that reside in several tissues, most abundantly in the small intestinal lamina propria under the steady state. To date, the phenotypic and functional characteristics of small intestinal eosinophils have remained poorly understood. In this study, we found that proliferation of ovalbumin (OVA)-specific CD4+ T cells isolated from the mesenteric lymph nodes of eosinophil-deficient DeltadblGATA mice were decreased relative to wild-type mice after oral immunization with OVA and cholera toxin (CT), the typical mucosal adjuvant that induces CD4+ T cell-dependent responses. DeltadblGATA mice showed reduced mucosal secretion of OVA-specific IgA and IgG1 while maintaining a systemic level of anti-OVA IgG1 upon oral immunization with OVA and CT. These findings suggest that eosinophils might have a role in the modulation of T cell-mediated immune responses including mucosal antibody responses in the gastrointestinal tract following oral immunization.

Imunogenicidade de proteínas do capsídeo do Cowpea severe mosaic virus (CPSMV) / Capsid protein immunogenicity of Cowpea severe mosaic virus (CPSMV)

A análise SDS-PAGE do Cowpea severe mosaic virus (CPSMV) purificado revelou a migração de três frações protéicas estimadas em 43, 23 e 21 kDa, correspondentes às proteínas do capsídeo: denominadas proteína maior (43 kDa) e menor (23 kDa; intacta e 21 kDa; clivada). As proteínas do capsídeo, na sua forma nativa, foram utilizadas na imunização de camundongos pelas vias oral e nasal, durante 10 dias consecutivos. As frações protéicas de 43 e 23 kDa, em sua forma desnaturada, foram utilizadas para imunização subcutânea. A resposta imunológica da mucosa foi avaliada pela proliferação celular das placas de Peyer de camundongos imunizados pela via oral com o CPSMV purificado. Ficou demonstrado que o CPSMV induz resposta imunológica, evidenciada pela síntese de anticorpos séricos, quando administrado na sua forma nativa pelas vias oral e nasal ou através de suas proteínas do capsídeo desnaturadas, pela via subcutânea. Não foi necessário o uso de adjuvantes, quer por via oral quer por via nasal. As frações protéicas de 43 e 23 kDa mostraram-se responsáveis pela imunogenicidade do vírus, como foi evidenciado pela síntese de anticorpos específicos detectados por ELISA. A análise da proliferação celular da placas de Peyer revelou um aumento (r=0,88) do número de leucócitos ao longo de 42 dias após a imunização. Esses resultados reforçam a possibilidade do uso do CPSMV como vetor seguro de antígenos de doenças humanas/animais pouco imunogênicos para produção de vacinas.

SDS-PAGE analysis of purified Cowpea severe mosaic virus (CPSMV) revealed the migration of three protein fractions of 43, 23 and 21 kDa, corresponding to the capsid protein called large protein (43 kDa) and small protein (23 kDa; intact and 21 kDa; cleaved). The capsid proteins, in their native form, were used to immunize mice through oral and nasal routes for ten consecutive days. The denatured form of the 43 and 23 kDa protein fractions were used for subcutaneous immunization. The mucosal immune response was detected by the cellular proliferation of the Peyer's patches of mice immunized by oral route with CPSMV. It was demonstrated that CPSMV induces immune response, evidenced by the synthesis of specific antibodies, when administered in the native form by the oral and nasal routes or with two denatured capsid proteins by the subcutaneous route. The use of adjuvants in the oral and nasal immunizations was not necessary. The 43 and 23 kDa protein fractions were responsible for the immunogenicity of the virus, evidenced by the synthesis of specific antibodies detected by ELISA test. The cellular proliferation analysis of the Peyer's patches revealed an increase (r=0.88) of leucocytes along 42 days after immunization. The results reinforce the possibility of the use of CPSMV as a safe vector of antigens for human/animal diseases of low immunogenicity for the production of vaccines.

Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety "Vitória de Verão" genetically modified.

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.

Influences of both genetic background and sex on the production of autoantibodies induced by vaccine of campylobacter jejuni / 中国免疫学杂志

Five strains of mice (C57BL/6, BALB/c, ICR, KM and SMMC/B) were respectively orally immunized with CJ-S131 vaccine for 16 weeks, Autoantibodies against ss-DNA, ds-DNA, histones, extractable nuclear antigens (ENA)and thymocytes were detected by ELISA or CELIS(?)One or more kind (s) of autoantibody significantly raised in the serum of SMMC/B, KM, BALB/c and ICR mice, but not in C57BL/6 mice. The mean enzyme index (EI) of the autoantibodies of SMMC/B and KM mice was higher than that of ICR and BALB/c. The influence of the sex on the production of autoantibody induced by CJ-S131 vaccine seemed to be demonstrated on the basis of genetic background.It was characterized by (1) autoantibodies raising mainly in the female mice,e.g. in KM mice; (2) one kind of autoantibody or more raising in both male and female mice, as seen in SMMC/B and BALB/c mice; and (3) autoantibody no in male and female of C57BL/6 mice. The results revealed that the profile of autoimmune response induced by CJ-S131 vaccine was different in strains and sex of mice.