Immunized Guinea Pigs Research Articles

A Novel Recombinant Virus-Like Particles Displaying B and T Cell Epitopes of Japanese Encephalitis Virus Offers Protective Immunity in Mice and Guinea Pigs.

Virus-like particles (VLPs) are non-replicative vectors for the delivery of heterologous epitopes and are considered one of the most potent inducers of cellular and humoral immune responses in mice and guinea pigs. In the present study, VLP-JEVe was constructed by the insertion of six Japanese encephalitis virus (JEV) envelope protein epitopes into different surface loop regions of PPV VP2 by the substitution of specific amino acid sequences without altering the assembly of the virus; subsequently, the protective efficacy of this VLP-JEVe was evaluated against JEV challenge in mice and guinea pigs. Mice immunized with the VLP-JEVe antigen developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. The neutralizing and hemagglutination inhibition (HI) antibody responses were also induced in guinea pigs vaccinated with VLP-JEVe. In addition, immunization with VLP-JEVe in mice induced effective neutralizing antibodies and protective immunity against PPV (porcine parvovirus) challenge in guinea pigs. These studies suggest that VLP-JEVe produced as described here could be a potential candidate for vaccine development.

The use of gene-modified bone marrow mesenchymal stem cells for cochlear cell therapy

BackgroundThe aim of this study was to investigate the potential of using bone marrow mesenchymal stem cells (BMSCs) for treatment of inflammation and autoimmune sensorineural hearing loss. MethodsFifty-five immunized guinea pigs were divided into five groups. Group A received BMSCs expressing IL-4, group B received BMSCs expressing an empty carrier vector, group C received recombinant lentivirus expressing IL-4, group D received recombinant lentivirus expressing an empty carrier vector, and group E received phosphate-buffered saline. Auditory function was monitored using brain stem responses (ABRs) to evaluate the auditory changes. The distribution of implanted BMSCs in the inner ear was estimated using fluorescence microscopy. The distribution and expression of IL-4 gene products in the inner ear were detected via immunohistochemistry. ResultsAfter transplantation, the ABR III wave threshold decreased significantly in BMSCs expressing exogenous IL-4 group (group A), BMSCs expressing empty carrier vector group (group B), and recombinant lentivirus expressing IL-4 group (group C) (P < 0.001), which means the auditory functions of the experimental guinea pigs were improved. Further statistical analysis revealed that BMSCs expressing exogenous IL-4 group (group A) and BMSCs expressing empty carrier vector group (group B) were able to improve the auditory function more obviously (P < 0.05). Lentivirus-infected BMSCs were able to migrate to the inner ear. Fluorescence-positive BMSCs were scattered in the scala tympani and vestibule. ConclusionsThese results demonstrated that BMSCs expressing exogenous IL-4 successfully migrated into the inner ear in an in vitro study. BMSCs expressing exogenous IL-4 and BMSCs can be used to treat inflammatory injury in autoimmune inner ear diseases.

A new candidate vaccine for human brucellosis based on influenza viral vectors: a preliminary investigation for the development of an immunization schedule in a guinea pig model

BackgroundA new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors (rIVV) subtypes H5N1 expressing Brucella outer membrane protein (Omp) 16, L7/L12, Omp19 or Cu–Zn SOD proteins has been developed. This paper presents the results of the study of protection of the vaccine using on guinea pigs, including various options of administering, dose and frequency. Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study.MethodsGeneral states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test. The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival, intranasal and sublingual administration in doses 105 EID50, 106 EID50 and 107 EID50 during prime and boost vaccinations of animals, followed by challenge with a virulent strain of B. melitensis 16 M infection. For sake of comparison, the commercial B. melitensis Rev.1 vaccine was used as a control. The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups.ResultsIt was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vaccine using conjunctival, intranasal and sublingual routes of administration, as well as after using various doses of vaccine. The most optimal way of using the vaccine has been established: double intranasal immunization of guinea pigs at a dose of 106 EID50, which provides 80% protection of guinea pigs from B. melitensis 16 M infection (P < 0.05), which is comparable to the results of the effectiveness of the commercial B. melitensis Rev.1 vaccine.ConclusionsWe developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model. We believe that because of these studies, the proposed vaccine has achieved the best level of protection, which in turn provides a basis for its further promotion.

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Comparison of Immunogenicity and Protective Efficacy of the Intranasal and Intraperitoneal Immunization Routes of &lt;i&gt;Escherichia albertii&lt;/i&gt; Strain DM104 in Mouse Model

In recent years, our group isolated the Escherichia albertii strain DM104 and characterized it as a vaccine strain against Shigella dysenteriae type 4 in the guinea pig eye model. Protective efficacy of different routes of immunization such as intranasal, oral, and intrarectal routes were also determined and compared by challenging immunized guinea pigs against live S. dysenteriae. In the current study, we compared the intranasal and intraperitoneal routes of immunizations with the DM104 vaccine strain in mice to understand the better route of administration of the DM104 vaccine and its immunogenicity as well as protective efficacy in mouse model. The results indicate that the immune response elicited by the DM104 strain is strongly dependent on the immunization route, with the intranasal route being more effective than the intraperitoneal route following intraperitoneal live S. dysenteriae challenge. Intranasal immunization yielded 80% protective efficacy in immunized mice whereas, intraperitoneal immunization could not provide any protection. Protection generated by intranasal immunization was accompanied by high titre of anti-whole cell lysate IgG and IgA in DM104 immunized sera compared to sera collected from mice of control group. All these data demonstrate the intranasal route of the vaccine DM104 strain in mouse model to be a better immunization route to protect the animals against live S. dysenteriae challenge.&#x0D; Bangladesh J Microbiol, Volume 37 Number 2 December 2020, pp 38-41

Preparation and optimization of rapid and sensitive coagglutination test for detection of infectious pancreatic necrosis virus (IPNV)

The aim of this study is the development of a coagglutination test for rapid diagnosis of infectious pancreatic necrosis (IPN) virus which causes an acute infectious viral disease with high mortality in young salmonid fish. Serotypes Sp, Ab, WB of the IPNV were cultivated in BF-2 cells and purified by linear sucrose density gradient centrifugation. Hyperimmune sera against purified IPNV serotypes were collected from immunized guinea pigs. Coagglutination test conjugates were prepared by using sensitized S. aureus and hyperimmune sera. Cell culture derived salmonid pathogens such as infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic hematopoietic necrosis virus (EHNV) were used to determine the specificity of the test as control. Upward comparison of the preferability for the rapid diagnosis was also carried out with ELISA and RT-PCR. The validation studies of newly developed coagglutination test were carried out in accordance with the Manual of Diagnostic Tests for Aquatic Animals. Consequently, determined detection limit of the test was approximately 105.25 TCID50/mL for the Sp and Ab serotypes and 107.75 TCID50/mL for the WB in laboratory conditions. The results indicated that newly developed coagglutination test was compatible with ELISA and RT-PCR in positive cell culture supernatants. It was concluded that this test would be an economic, rapid and reliable method for the diagnosis of IPN virus in cell culture.

Convalescent Immunity to Guinea Pig Cytomegalovirus Induces Limited Cross Strain Protection against Re-Infection but High-Level Protection against Congenital Disease.

The guinea pig is the only small animal model for congenital cytomegalovirus (cCMV) but requires guinea pig cytomegalovirus (GPCMV). Current GPCMV research utilizes prototype strain 22122, which limits the translational impact of GPCMV as numerous human CMV strains exist and cCMV is possible in the setting of re-infection. A novel strain of GPCMV (TAMYC) exhibited differences to 22122 in various glycoproteins with GP74 (gO homolog) the most variable (25% difference). Antibody ELISAs for TAMYC-convalescent animals evoked similar immune response to viral glycoprotein complexes (gB, gH/gL, gM/gN, pentamer) and cell-mediated response to pp65 homolog (GP83). Convalescent sera from TAMYC-infected animals neutralized GPCMV infection on fibroblasts but was less effective on epithelial cells. TAMYC-convalescent animals were not protected from dissemination of heterogenous virus challenge (22122). However, in a cCMV protection study, TAMYC-convalescent animals challenged mid-pregnancy (22122) exhibited high-level protection against cCMV compared to seronegative animals with pup transmission reduced from 80% (control) to 12%. Overall, pre-existing immunity in guinea pigs provides limited ability to prevent GPCMV re-infection by a different viral strain but provides a high level of protection against cCMV in heterogenous strain challenge. This level of cross protection against cCMV should be a prerequisite of any CMV vaccine.

Protection against Borreliella burgdorferi infection mediated by a synthetically engineered DNA vaccine

ABSTRACT Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.

Adenovirus-Vectored Capsid Proteins of the Serotype A Foot-and-Mouth Disease Virus Protect Guinea Pigs Against Challenge.

Type A foot-and-mouth disease virus (FMDV) has been detected on China’s pig farms since 2015, and all suspected samples have been strain A/GDMM/CHA/2013. To overcome the shortcomings of inactive FMDV vaccines, we expressed the capsid protein precursor P1-2A and mutated viral 3C protease of FMDV strain A/GDMM/CHA/2013 in a replication-deficient human adenovirus type 5 vector in this study. A significant humoral immune response, T-cell-mediated antiviral response, and mucosa-mediated antiviral response were induced by the adenovirus-vectored FMDV vaccines in BALB/c mice. Immunization of guinea pigs with the adenovirus-vectored FMD vaccines induced significant neutralizing antibodies and anti-FMDV immunoglobulin A antibodies. The recombinant adenovirus rAdv-P12A3CG38SF48S-GD protected 100% of guinea pigs against challenge when administered intramuscularly. Our study demonstrated the potential utility of rAdv-P12A3CG38SF48S-GD as a vaccine against type A FMDV.

Immunogenicity of a DNA vaccine candidate for COVID-19

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

Elicitation of Cluster A and Co-Receptor Binding Site Antibodies are Required to Eliminate HIV-1 Infected Cells.

HIV-1-infected individuals raise a polyclonal antibody response targeting multiple envelope glycoprotein (Env) epitopes. Interestingly, two classes of non-neutralizing CD4-induced (CD4i) antibodies, present in the majority of HIV-1-infected individuals have been described to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of small CD4 mimetic compounds (CD4mc). These antibodies recognize the coreceptor binding site (CoRBS) and the constant region one and two (C1C2 or inner domain cluster A) of the gp120. In combination with CD4mc they have been shown to stabilize an antibody-vulnerable Env conformation, known as State 2A. Here we evaluated the importance of these two families of Abs in ADCC responses by immunizing guinea pigs with gp120 immunogens that have been modified to elicit or not these types of antibodies. Underlying the importance of anti-CoRBS and anti-cluster A Abs in stabilizing State 2A, ADCC responses were only observed in the presence of these two types of CD4i antibodies. Altogether, our results suggest that these two families of CD4i antibodies must be taken into account when considering future strategies relying on the use of CD4mc to eliminate HIV-1-infected cells in vivo.

Evaluation of allergenic properties and immunotoxicity of Homeovox

Homeovox, a multicomponent homeopathic treatment for laryngitis of various etiologies, was tested for allergenic properties and immunotoxicity.Methods. 80 male albino guinea pigs and 180 male СВА, C57BL/6 and F1 hybrid (CBAхС57BL/6) mice were used in the study. In order to assess immunotoxicity, Homeovox was administrated orally to mice for 14 days at doses of 100 mg/kg and 1 000 mg/kg. To assess the allergenic properties of Homeovox, albino guinea pigs were also given the drug at doses of 100 mg/kg and 1 000 mg/kg according to the standard immunization schedules.Results. Oral administration of Homeovox to mice for 14 days at doses of 100 mg/kg and 1 000 mg/kg did not cause significant changes in the main characteristics of immune response compared to controls. When assessing allergenicity, Homeovox at doses 100 mg/kg and 1 000 mg/kg administered according to the standard immunization schedules did not induce systemic anaphylaxis or active skin anaphylaxis in albino guinea pigs. The immunization of guinea pigs by Homeovox at the same doses in a mixture with Freund's complete adjuvant caused no delayed allergic reactions. Homeovox at a single oral dose of 1 000 mg/kg significantly decreased concanavalin A-induced inflammation in CBA mice by 58.4 %.Conclusion. Within the dosage range investigated, Homeovox does not induce immunotoxicity, immediate- or delayed-type allergic or pseudoallergic reactions.

A Lassa Virus Live-Attenuated Vaccine Candidate Based on Rearrangement of the Intergenic Region.

Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosycases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Comparison of immune responses in guinea pigs by intranasal delivery with different nanoparticles-loaded FMDV DNA vaccine

To compare different nanoparticle-based nasal vaccines against foot-and-mouth disease (FMD), chitosan (CS)-coated poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) (CS/PLGA-NPs) and amino-functionalized mesoporous silica nanoparticles (Am/MSNs) loaded with FMDV recombinant plasmid (pP12A3C/IFN-CS/PLGA-NPs and pP12A3C/IFN-Am/MS-NPs) were used to induce mucosal and systemic immune responses in guinea pigs via intranasal delivery. Simultaneously, CpG oligodeoxy nucleotides (ODNs) as a vaccine adjuvant were encapsulated in chitosan-coated poly (lactic-co-glycolic acid) nanoparticles (CpG-CS/PLGA-NPs). The pP12A3C/IFN-CS/PLGA-NPs and CpG-CS/PLGA-NPs generated displayed good morphology, high stability, mean diameters of 500 and 400 nm and encapsulation efficiencies of 83.8% and 88.4%, respectively. Data from the in vitro release assay showed that plasmid and CpG were sustainably released from nanoparticles (up to 66.73% and 64%, respectively, of the total amount loaded). Guinea pigs immunized with pP12A3C/IFN-CS/PLGA-NPs + CpG-CS/PLGA-NPs showed markedly higher mucosal, cellular and humoral immune responses than those administered pP12A3C/IFN-CS/PLGA-NPs or naked plasmid vaccine alone. FMDV-specific secretory immunoglobulin A (sIgA) antibodies in nasal washes were initially detected at 3 days post-vaccination with CS/PLGA-NPs loaded with plasmid. Guinea pigs immunized with pP12A3C/IFN-CS/PLGA-NPs also displayed higher cellular and humoral immune responses than pP12A3C-CS/PLGA-NPs and naked plasmid vaccine alone. FMDV-specific immunoglobulin G (IgG) antibodies in serum were initially detected at 5 days post-vaccination (intramuscularly) with the naked plasmid. Finally, challenge experiments 42 days post-vaccine revealed 100% protection in guinea pigs immunized with pP12A3C/IFN-CS/PLGA-NPs + CpG-CS/PLGA-NPs and pP12A3C/IFN-CS/PLGA-NPs. However, plasmid DNA was burst released from pP12A3C/IFN-Am/MS-NPs. Our attempts to use pP12A3C/IFN-Am/MS-NPs to immunize guinea pigs failed to induce immune responses. In conclusion, CpG and IFN-α adjuvant based FMD vaccines elicit protection in guinea pigs. Moreover, CS-coated PLGA NPs present an efficient and safe mucosal immune delivery system for FMDV DNA vaccine. Data from the current study provide a foundation for understanding and further evaluating protective immune responses in pigs.

Immunogenicity and protective efficacy of 3A truncated negative marker foot-and-mouth disease virus serotype A vaccine

Foot-and-mouth disease (FMD) is a highly contagious, economically significant disease of cloven-hoofed animals caused by FMD virus (FMDV) of the Picornaviridae family. Vaccination of susceptible animals with inactivated virus vaccine is the standard practice for disease control. The prophylactic use of the inactivated vaccines has reduced the disease burden in many countries endemic to FMD. In the process of implementation of the mass vaccination program and disease eradication, it is essential to differentiate infected from vaccinated animals (DIVA) where a large proportion of the animal population is vaccinated, and disease-free zones are being established, to help in sero-surveillance of the disease. In such a scenario, the use of a negative marker vaccine is beneficial to rule out false-positive results in a disease-free zone. Here we report the construction and rescue of an infectious cDNA clone for FMDV serotype A Indian vaccine strain lacking 58 amino acid residues (87-144 amino acid position) in the carboxy-terminal region of the viral 3A protein. The recombinant deletion mutant virus showed similarity in the antigenic relationship with the parental strain. Immunization of guinea pigs with the inactivated vaccine formulated using the deletion mutant virus induced potent immune response with 100% protective efficacy upon challenge with homologous virus. Further, we show that sera from the guinea pigs infected with the deletion mutant virus did not show reactivity in an indirect ELISA test targeting the deleted portion of 3A protein. We conclude that the recombinant deletion mutant virus vaccine along with the newly developed companion indirect ELISA targeting portion of FMDV 3A protein could be useful in the implementation of a precise DIVA policy in our country when we reach FMD free status with vaccination.

EXPERIMENTAL MODELING OF MULTIPLE SCLEROSIS: THE RESULTS OF CORRECTIVE ACTION OF ACTIVE ANTIOXIDANT

Comprehensive treatment of patients with multiple sclerosis (MS) is still relevant. The complex pathogenesis and acute incidence of MS in young people requires the use of multifunctional drugs with a protective effect, including antioxidants. The purpose of this study is to identify a corrective neuroprotective and immune modulating effect of the synthetic drug Triafen-2 (Trf-2), obtained in optimized way. Trf-2 was used at a dose of 50 mg/kg orally with chronic administration in II parts of experiments. To create an adequate model of MS - autoimmune encephalomyelitis (EAE), immunization of guinea pigs with an encephalitic mixture was used in Freund’s complete adjuvant. Mortality, a number of immunological parameters, the clinical picture of neurological complications and the severity of CNS damage in guinea pigs were determined. We studied morphologically the development of T-lymphocytic infiltration of the brain and spinal cord, demyelination of nerve fibers, the degree of cerebellar disorders, disorders of the pelvic organs, the presence of paresis and paralysis. A significant decrease in mortality was shown and protective effect of Trf-2 on neurological symptoms of EAE was noted. The reduction and even complete absence of paresis and paralysis in 66.5% of guinea pigs in the experimental group compared with animals that did not receive treatment was determined. A positive effect of Trf-2 on the migration activity of leukocytes and antibodies in the blood was found. In the group with Trf-2, there was a decrease in the content of lipid hydroperoxides and malonic dialdehyde. A pronounced neuroprotective and immune modulating effect of Triafen-2, which inhibits pathological disorders in an experimental model of EAE, has been established. The results justify the need for further study of Triafen-2 as a promising corrective drug.

Dynamic analysis of antibodies induced by leptospiral vaccines

Objective To investigate the dynamic changes of antibodies induced by leptospiral vaccines. Methods Antigens for antibody detection were screened out. ELISA was used to analyze antibody responses induced at different time points after immunizing guinea pigs with different batches of leptospiral vaccines from different manufacturers. To investigate the relationship between antibody responses induced by leptospiral vaccines and their protective effects in animal model, guinea pigs were challenged with Leptospira after immunization. Results There was no significant antigen-antibody reaction between the LigA protein or Patoc Ⅰ antigen and the serum samples of guinea pigs immunized with leptospiral vaccines. Notable IgG and IgM antibody reactions were observed in all vaccination groups when using bacterial proteins from seven Leptospira reference strains which were used for the preparation of leptospiral vaccines as envelope antigens. Antigen-specific IgG antibodies peaked at 35 d after the last immunization, and the highest peak of antigen-specific IgM antibodies was reached 11 d after the last immunization. Results of the challenge test showed that non-diluted leptospiral vaccines induced significant IgG and IgM antibody reactions in guinea pigs as compared with those diluted three or nine times, showing good protective effects. Conclusions Analysis of the dynamic changes of antibodies induced by leptospiral vaccines revealed that there was correlation between the induced serum antibody responses and the protective effects. This study provided reference for further study on alternative methods for evaluating leptospiral vaccine potency. Key words: Leptospira; Vaccine; Antibody; Protection

Soluble FMDV VP1 proteins fused with calreticulin expressed in Escherichia coli under the assist of trigger factor16 (Tf16) formed into high immunogenic polymers

Foot and mouth disease virus (FMDV) is a highly contagious pathogen propagating among cloven-hoofed animals. As a major immunogenic protein, VP1 plays a pivotal role in the induction of neutralizing antibodies, which therefore is an ideal target for developing subunit vaccines. In current study, four prokaryotic expression clones (rV4C, rC4V, rV5F and rF5V) were constructed by fusing truncated calreticulin (CRT) (120–250 aa or 120–308 aa) at the N/C terminal of vp1 gene, and co-expressed with chaperone trigger factor 16 (Tf16) in E.coli, respectively. The soluble recombinant CRT-fused VP1 proteins could form into homogeneous reactive polymers with average hydrodynamic diameters around 100 nm according to the dynamic light scattering (DLS) data. Immunization of guinea pigs with 10 μg purified CRT-fused VP1 proteins induced high levels of antibodies against naked-VP1 through indirect ELISA. Sandwich ELISA showed that only rC4V could elicit the same level of antibody against FMD virus as commercial inactivated vaccine after booster. The lymphocyte cytokines secretion of immunized rC4V was higher than the other CRT-fused VP1 proteins in guinea pigs. These results showed that the soluble CRT-fused VP1 proteins, especially rC4V, expressed with Tf16 in E. coli might have potential to be used as subunit vaccine candidate against FMDV.

Intranasal nanoemulsion-adjuvanted HSV-2 subunit vaccine is effective as a prophylactic and therapeutic vaccine using the guinea pig model of genital herpes

Genital herpes is a sexually transmitted disease representing a major global health concern. Currently, there is no approved vaccine and existing antiviral therapies exhibit limited efficacy. Herein, we describe an intranasal (IN) vaccine comprised of HSV-2 surface glycoproteins gD2 and gB2 formulated in a nanoemulsion adjuvant (NE01-gD2/gB2). Using the HSV-2 genital herpes guinea pig model, we demonstrate that IN NE01-gD2/gB2 induces higher levels of neutralizing antibody compared to a monovalent IN NE01-gD2 vaccine, but less than an intramuscular (IM) Alum/MPL-gD2 vaccine. Following intravaginal (IVag) challenge with HSV-2, the group immunized with IN NE01-gD2/gB2 exhibited significantly reduced acute and recurrent disease scores compared to placebo recipients. Significantly, latent virus was only detected in the dorsal root ganglia of 1 of 12 IN NE01-gD2/gB2-vaccinated animals compared to 11 of 12 placebo recipient. In the therapeutic model, IN NE01-gD2/gB2 immunized guinea pigs exhibited a significant reduction in the recurrent lesions scores (64%, p < 0.01), number of animal days with disease (64%, p < 0.01), number of animals with viral shedding (50%, p < 0.04) and reduction in virus positive vaginal swabs (56%, p < 0.04), These data suggests that the treatment may be effective in treating chronic disease and minimizing virus transmission. These results warrant advancing the development of IN NE01-gD2/gB2 as both a prophylactic and therapeutic vaccine against HSV-2.