Triple Immunization Research Articles

Screening and anti-angiogenesis activity of Chiloscyllium plagiosum anti-human VEGFR2 single-domain antibody.

Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (variable new antigen receptor, VNARs), was considered to have the high antigen-binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6, and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized C. plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs.

Analysis of Immunobiological Properties of Recombinant Streptococcus pneumoniae Pneumolysin.

We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH)3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH)3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH)3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection.

Study of the safety and immunogenicity of VLP-based vaccine for the prevention of rotavirus infection in neonatal minipig model

In Russia, almost half of the cases of acute intestinal infections of established etiology in 2022 are due to rotavirus infection (RVI). There is no specific treatment for rotavirus gastroenteritis. There is a need to develop modern, effective and safe vaccines to combat rotavirus infection that are not capable of multiplying (replicating) in the body of the vaccinated person. A promising approach is to create vaccines based on virus-like particles (VLPs). Study of the safety and immunogenicity of a vaccine against rotavirus infection based on virus-like particles of human rotavirus A in newborn minipigs with multiple intramuscular administration. Newborn minipigs were used as an animal model in this study. The safety of the tested vaccine was assessed based on thermometry data, clinical examination, body weight gain, clinical and biochemical blood parameters, as well as necropsy and histological examination. When studying the immunogenic properties of the Gam-VLP-rota vaccine in doses of 30 and 120 µg, the cellular, humoral and secretory immune response was studied. The results of assessing the general condition of animals during the immunization period, data from clinical, laboratory and pathomorphological studies indicate the safety of the vaccine against human rotavirus infection based on VLP (Gam-VLP-rota) when administered three times intramuscularly. Good local tolerance of the tested vaccine was demonstrated. The results of the assessment of humoral immunity indicate the formation of a stable immune response after three-time immunization with Gam-VLP-rota, stimulation of the production of antigen-specific IgG antibodies and their functional activity to neutralize human rotavirus A. It was shown that following the triple immunization with the minimum tested concentration of 30 µg/dose, animals developed a cell-mediated immune response. The results of the IgA titer in blood serum and intestinal lavages indicate the formation of both a systemic immunological response and the formation of specific secretory immunity to human rotavirus A. Thus, three-time intramuscular immunization of minipigs with the Gam-VLP-rota vaccine forms stable protective humoral and cellular immunity in experimental animals. Evaluated vaccine is safe and has good local tolerability.

Preparation of a Klebsiella pneumoniae conjugate nanovaccine using glycol-engineered Escherichia coli

BackgroundEngineered strains of Escherichia coli have been used to produce bioconjugate vaccines using Protein Glycan Coupling Technology (PGCT). Nanovaccines have also entered the vaccine development arena with advances in nanotechnology and have been significantly developed, but chassis cells for conjugate nanovaccines have not been reported.ResultsTo facilitate nanovaccine preparation, a generic recombinant protein (SpyCather4573) was used as the acceptor protein for O-linked glycosyltransferase PglL, and a glycol-engineered Escherichia coli strain with these two key components (SC4573 and PglL) integrated in its genome was developed in this study. The targeted glycoproteins with antigenic polysaccharides produced by our bacterial chassis can be spontaneously bound to proteinous nanocarriers with surface exposed SpyTag in vitro to form conjugate nanovaccines. To improve the yields of the targeted glycoprotein, a series of gene cluster deletion experiments was carried out, and the results showed that the deletion of the yfdGHI gene cluster increased the expression of glycoproteins. Using the updated system, to the best of our knowledge, we report for the first time the successful preparation of an effective Klebsiella pneumoniae O1 conjugate nanovaccine (KPO1-VLP), with antibody titers between 4 and 5 (Log10) after triple immunization and up to 100% protection against virulent strain challenge.ConclusionsOur results define a convenient and reliable framework for bacterial glycoprotein vaccine preparation that is flexible and versatile, and the genomic stability of the engineered chassis cells promises a wide range of applications for biosynthetic glycobiology research.

Serotype-independent protective activity of recombinant pneumolysin in a model of systemic infection caused by &lt;i&gt;Streptococcus pneumoniae&lt;/i&gt; serotypes 4 and 6B

Background. The development of a pneumococcal vaccine with serotype-independent activity is relevant for the whole world, as this is due to the high prevalence of Streptococcus pneumoniae (S. pneumoniae), the constant change of pathogen serotypes and the growth of antibiotic-resistant strains. In previous experiments, we revealed a protective effect of immunization with recombinant pneumolysin (rPly) in a model of systemic infection caused by S. pneumoniae serotype 3.Aim. Study of serotype-independent protective activity of rPly.Materials and methods. Strains of S. pneumoniae serotypes 4 and 6B were used. Mice were immunized intraperi-toneally two or three times with an interval of 14 days with rPly. To assess the protective activity of rPly, animals after double or triple immunization were infected intraperitoneally with S. pneumoniae .Results. rPly at a triple immunization in a dose of 25 μg protected mice from intraperitoneal infection with S. pneu- moniae serotype 4, the efficiency index increased by 2 times compared with the control. with a double immunization, rPly protected mice from intraperitoneal infection with S. pneumoniae serotype 6B, efficiency index increased by 6.61 times compared with the control.Conclusion. rPly protects animals from infection with different serotypes of S. pneumoniae, which allows us to consider it a promising drug for the development of pneumococcal vaccines with serotype-independent activity.

Longitudinal monitoring of post-vaccination immunity against hepatitis B in children after standard immunization in the first year of life and optimization of vaccination strategy

A clinical and laboratory examination of 62 children aged 10—17 years vaccinated against hepatitis B according to the standard scheme (0—1—6 months) in the first year of life strictly according to the National Calendar of preventive vaccinations in a standard dose (10 micrograms of recombinant HBsAg) with a monocomponent vaccine was carried out.The majority of children (58%) had indicators of seroconversion of HBsAg — anti-HBs-antibodies. This indicates the fact of long-lasting Grund immunity — 10—17 years after timely 3-fold immunization against hepatitis B according to the standard scheme at a standard dose (10 mcg of recombinant HBsAg in the first year of life in the absence of Wooster immunization (revaccination) in subsequent age periods, including children with a burdened premorbid background (87.1% among those observed). Since among those observed were children with invasive medical interventions (50%), the established fact reflects the protective effect of standard Grund immunization even in children at risk — with a positive parenteral history. At the same time, the total assessment of serological and molecular biological results found that after triple immunization against hepatitis B according to the standard scheme in the first year of life, out of 62 children aged 10—17 years, the proportion of seronegative was 42%. Low levels of antibodies (10—100 IU/l) were detected in 43% of cases, high (100—1000 IU/l) — in 15% of cases. No child had an antibody level exceeding &gt;1000 IU/l. These results require the development of new approaches to immunization of children against hepatitis B with mandatory revaccination at an older age.

Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern.

Infection-neutralizing antibody responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 vaccination are an essential component of antiviral immunity. Antibody-mediated protection is challenged by the emergence of SARS-CoV-2 variants of concern (VoCs) with immune escape properties, such as omicron (B.1.1.529), which is rapidly spreading worldwide. Here we report neutralizing antibody dynamics in a longitudinal cohort of coronavirus disease 2019 convalescent and infection-naive individuals vaccinated with mRNA BNT162b2 by quantifying SARS-CoV-2 spike protein antibodies and determining their avidity and neutralization capacity in serum. Using live-virus neutralization assays, we show that a superior infection-neutralizing capacity against all VoCs, including omicron, developed after either two vaccinations in convalescents or a third vaccination or breakthrough infection of twice-vaccinated, naive individuals. These three consecutive spike antigen exposures resulted in an increasing neutralization capacity per anti-spike antibody unit and were paralleled by stepwise increases in antibody avidity. We conclude that an infection-plus-vaccination-induced hybrid immunity or a triple immunization can induce high-quality antibodies with superior neutralization capacity against VoCs, including omicron.

Influence of Caudovirales Phages on Humoral Immunity in Mice.

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Experimental Protein-Containing Preparations &lt;i&gt;Streptococcus pneumoniae&lt;/i&gt;, Obtained from Fresh Isolated Strains and Museum

Relevance. Vaccines based on capsular polysaccharides of pneumococci are not active against serotypes that are not included in the vaccine, non-capsulated strains and do not protect against carriage caused by other serotypes. Their use leads to the replacement of dominant serotypes of pneumococci, the appearance of highly virulent strains, changes in the microbial landscape of mucous membranes due to the appearance of other etiologically significant pathogens of respiratory tract diseases. This requires the creation of intraspecific anti-pneumococcal immunity, which will be facilitated by the development of serotype-dependent drugs, which will include protein-containing antigens of pneumococci. Objective. Study of serotype-independent activity of protein-containing antigenic components obtained from freshly isolated and archival strains of S. pneumoniae. Materials and methods. Strains of three serotypes of S. pneumoniae were used: archival strains of serotypes 6B N296, 19F N 298 and 10A N297 and freshly isolated serotypes 6B N 1121, 19F N 1055 and serogroup 10 N 1193 (from the cerebrospinal fluid of patients with purulent meningitis).. In the experimental protein-containing preparations EPCP obtained, the protein content was determined. Protective activity of EPCP and virulence of strains were determined in the model of intraperitoneal immunization and infection of BALB/c mice; LD50 was calculated using the generally accepted modified Kerber formula. The immunophenotype of lymphocytes previously isolated from donor-mice whole blood was studied by flow cytometry. Statistical analysis of the materials was carried out using parametric and nonparametric methods using the application package «Statistica for Windows», ver. 7.0 (Stat Soft, Inc); in statistical analysis, the significance level of p was assumed to be &lt; 0.05. Results and discussion. During cultivation there was the analysis of growth dynamics showed intensive accumulation of biomass of the archival strain N296 with low virulence, and lower - by virulent strain N 1121 isolated from the patient's cerebrospinal fluid. The protein content of drugs from serotype 6B strains did not differ. The strains of other serotypes isolated during the generalized infectious process were also more virulent than the archival strain. The effect on the immunophenotype of human lymphocytes of fractions of 50-100 kDa isolated from the initial preparations obtained by culturing serotype 6B, significantly increased only under the influence of the preparation from the archival strain. In the study of protective activity of the initial preparations from strains of serotype 6B and fractions with MM 50-100 kDa only triple immunization with the initial preparation from strain N 296, at a dose of 20 micrograms of protein per mouse, led to significantly greater survival of immunized mice from infection with the newly isolated virulent strain N 1121 of homologous serotype. A fraction of 30-100 kDa provided protection of mice twice immunized with 50 mkg of protein per mouse, with a high efficacy index of 8.9, even after infection with a freshly isolated strain of heterologous S. pneumoniae serotype 3 N 10196. Conclusion. The protein-containing fraction with MM 30-100 kDa obtained from a low virulent archival strain of S. pneumoniae serotype 6B N296 possessed protective activity against a newly isolated virulent strain of heterologous serotype after double immunization. Under the action of the studied protein-containing fractions, activation of the cellular component of immune system with the involvement of innate immunity effectors and T-lymphocytes is shown. These data can be considered as a evidence for further study of EPCP to assess the possibility of their use in the design of anti-pneumococcal drug with serotype-independent protective activity.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle–immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Hollow microneedle-mediated intradermal delivery of model vaccine antigen-loaded PLGA nanoparticles elicits protective T cell-mediated immunity to an intracellular bacterium

The skin is an attractive organ for immunization due to the presence of a large number of epidermal and dermal antigen-presenting cells. Hollow microneedles allow for precise and non-invasive intradermal delivery of vaccines. In this study, ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with and without TLR3 agonist poly(I:C) were prepared and administered intradermally by hollow microneedles. The capacity of the PLGA nanoparticles to induce a cytotoxic T cell response, contributing to protection against intracellular pathogens, was examined. We show that a single injection of OVA-loaded PLGA nanoparticles, compared to soluble OVA, primed both adoptively transferred antigen-specific naïve transgenic CD8+ and CD4+ T cells with markedly high efficiency. Applying a triple immunization protocol, PLGA nanoparticles primed also endogenous OVA-specific CD8+ T cells. Immune response, following immunization with in particular anionic PLGA nanoparticles co-encapsulated with OVA and poly(I:C), provided protection against a recombinant strain of the intracellular bacterium Listeria monocytogenes, secreting OVA. Taken together, we show that PLGA nanoparticle formulation is an excellent delivery system for protein antigen into the skin and that protective cellular immune responses can be induced using hollow microneedles for intradermal immunizations.

Comparing New-Generation Candidate Vaccines against Human Orthopoxvirus Infections

The lack of immunity to the variola virus in the population, increasingly more frequent cases of human orthopoxvirus infection, and increased risk of the use of the variola virus (VARV) as a bioterrorism agent call for the development of modern, safe vaccines against orthopoxvirus infections. We previously developed a polyvalent DNA vaccine based on five VARV antigens and an attenuated variant of the vaccinia virus (VACV) with targeted deletion of six genes (VACΔ6). Independent experiments demonstrated that triple immunization with a DNA vaccine and double immunization with VACΔ6 provide protection to mice against a lethal dose (10 LD50) of the ectromelia virus (ECTV), which is highly pathogenic for mice. The present work was aimed at comparing the immunity to smallpox generated by various immunization protocols using the DNA vaccine and VACΔ6. It has been established that immunization of mice with a polyvalent DNA vaccine, followed by boosting with recombinant VACΔ6, as well as double immunization with VACΔ6, induces production of VACV-neutralizing antibodies and provides protection to mice against a 150 LD50 dose of ECTV. The proposed immunization protocols can be used to develop safe vaccination strategies against smallpox and other human orthopoxvirus infections.

The Study of Protective Properties of Vaccines against Virulent Strains of the Virus Tick-Borne Encephalitis Three Genotypes: European, Far Eastern and Siberian (Experimental Research)

Researches and presents comparative characteristics of protective properties of vaccines EC domestic and foreign production by immunization line BALB/c mice in a standard experiment for estimating the immunogenic activity against virulent strains of TBE virus three genotypes: genotype 1 - the strain «Sof'in», genotype 2 - strain «Absettarov», genotype 3 - strain «Lesopark». The results are of considerable interest in the evaluation of the efficiency and interchangeability of vaccines for the prevention of TBE in areas of natural foci of the disease in Russia. Currently, the circulation of the pathogen in the Russian Federation dominated by the TBE virus strains Siberian subtype (genotype) -more than 70% of virulent strains of TBE virus released into the natural foci of the Urals, Siberia, the Trans-Baikal. In studies on mice of BALB/c did not reveal significant differences in terms of protection (MID50 and DT50) against virulent strains of TBE virus three genotypes - the Far East, Europe and Siberia for three vaccines: «The vaccine encephalitis culture purified concentrated dry» (production Mikhail Chumakov Federal Research Centre for Research and Development of Immunobiological Preparations, Moscow), EntseVir (production Federal State Unitary Enterprise «Scientific and Production Association «Microgen»), FSME-Immun (Baxter AG production, Austria). Vaccines have shown good protection (protective) properties against 3 genotypes of TBE virus. Revealed a lower protective activity of vaccines Entsepur (manufactured by Novartis Vaccines and Diagnostics GmbH & Co. KG, Germany) strains of TBE 1 and 3 genotypes. These results confirm earlier studies to study the humoral response in mice BALB/c in the experiments with double and triple immunization.

Assessment of Protective Properties of the Recombinant Complex of the Outer Membrane Protein F and the Toxoid of Pseudomonas aeruginosa

Pseudomonas aeruginosa induces the complications after burns, injuries, surgical interventions and appears to be one of the main causative agents of nosocomial infections. This pathogen has the high resistance to the antibacterial preparations, therefore the immunoprophylaxis is considered as one of the major approaches to reduce Pseudomonas infection. Objective: The aim of our investigation is to study the protective properties of the recombinant complex of the outer membrane protein F (OprF) and a non-toxic variant of the exotoxin A (toxoid) against Pseudomonas infection. Methods: The recombinant proteins which contained the additional histidine residues were synthesized into Escherichia coli with isopropyl-βD-thyogalactopyranoside (IPTG). The recombinant proteins were purified by affinity chromatography on Ni-Sepharose. The preparations of recombinant proteins were injected intraperitoneally into the mice. Aluminum hydroxide was used as an adjuvant. For an experimental infection in mice, animals were challenged intraperitoneally by a live virulent culture of P. aeruginosa (PA-103 strain). Results: The best protective effect for the complex containing 25 μg OprF and 50 μg toxoid was identified when we used the double immunization of mice (Index of efficiency of the protective properties in this case was 4.0). Indexes of efficiency of separated recombinant proteins which were injected twice in the same doses were 2.0 for OprF и 2.3 for toxoid. The triple immunization of animals was inefficient for separated recombinant proteins in the same doses. The injection of doses which were lowered twice (12.5 μg for OprF and 25 μg for toxoid) resulted in increased survival of mice immunized by individual proteins (indexes of efficiency: 3 for OprF and и 3,5 for toxoid). However when we administered to the complex of proteins with the same doses Index of efficiency was 2.8. Conclusion: It was shown that the maximum protective effect in a short time is achieved by the combination of double immunization and the mixture of the recombinant proteins OprF and the 25 and 50 μg doses of recombinant toxoid .

FORMATION OF INNATE AND ADAPTIVE IMMUNE RESPONSE UNDER THE INFLUENCE OF DIFFERENT FLAVIVIRUS VACCINES

The review examines in a comparative perspective the key moments of formation of innate and adaptive immune responses to different types of current flavivirus vaccines: live attenuated against yellow fever virus and inactivated whole virus against tick-borne encephalitis virus. Particular attention is paid to the ability of these different vaccines, containing exogenous pathogen-associated molecular structures, to stimulate innate immunity. Live attenuated vaccine by infecting several subtypes of dendritic cells activates them through various pattern-recognition receptors, such as Tolland RIG-I-like receptors, which leads to significant production of proinflammatory cytokines, including interferon-α primary mediator of innate antiviral immunity. By simulating natural viral infection, this vaccine quickly spreads over the vascular network, and the dendritic cells, activated by it, migrate to the draining lymph nodes and trigger multiple foci of Tand B-cell activation. Inactivated vaccine stimulates the innate immunity predominantly at the injection site, and for the sufficient activation requires the presence in its composition of an adjuvant (aluminum hydroxide), which effects the formation and activation of inflammasomes, ensuring the formation and secretion of IL-1β and IL-18 that, in turn, trigger a cascade of cellular and humoral innate immune responses. We demonstrated the possibility of involvement in the induction of innate immunity, mediated by the inactivated vaccine, endogenous pathogenassociated molecular patterns (uric acid and host cell DNA), forming at the vaccine injection site. We discuss the triggering of Band T-cell responses by flavivirus vaccines that determine various duration of protection against various pathogens. A single injection of the live vaccine against yellow fever virus induces polyvalent adaptive immune response, including the production of cytotoxic T-lymphocytes, Th1and Th2-cells and neutralizing antibodies, which may persist for up to 40 years after the vaccination. To induce and maintain protective immunity, mediated by the inactivated vaccine against tick-borne encephalitis virus, it is required: triple immunization, which results in the production primarily of neutralizing antibodies, and subsequent booster injections every 3 years. We considered the potential use of the data on immunological mechanisms of action of current vaccines to generate new highly efficient vaccines.

Enhancement of the immune response by codelivery of hepatitis C virus recombinant DNA and proteins of the replicative complex

The plasmids encoding amino acid sequences corresponding to the hepatitis C virus (HCV) are regarded as promising candidates for anti-HCV vaccines. However, the optimal composition of these vaccines has not been determined. The goal of this work was to evaluate the immune response after immunization of mice with the DNA construct pcNS3-NS5B encoding proteins that constitute the viral replicative complex combined with the recombinant nonstructural proteins NS3 and NS5B using various adjuvants. DBA mice were immunized with the DNA construct and/or recombinant proteins three times. The antibody response was evaluated using ELISA. The cell immunity was determined using a lymphocyte proliferation test in vitro and by production and secretion of IFNγ and IL-2 measured by ELISpot and ELISA. It was found that the pcNS3-NS5B plasmid induced predominantly T-cell response, whereas the recombinant NS3 and NS5B proteins stimulated a potent humoral immune response. This made it possible for us to show for the first time statistically significant stimulation of both humoral and cell immunity after immunization with the pcNS3-NS5B plasmid in combination with the NS3 and NS5B HCV proteins. It was noteworthy that the animals developed the immune response not only to the NS3 and NS5B proteins, but also to other antigens encoded by the pcNS3-NS5B plasmid (NS4, NS5A). The most pronounced immune response was observed in mice subjected to triple immunization with the pcNS3-NS5B plasmid in combination with the gene adjuvant pcGM-CSF and recombinant proteins (NS3 and NS5B), which were injected together with IFNα. The adjuvant activity of the recombinant protein IFNα, which was shown in a model of the HCV genes and proteins for the first time, may be a prerequisite for including it in a candidate vaccine.

Evaluation of systemic and mucosal immune responses in mice administered with recombinant Salmonella Typhimurium expressing IutA protein

Abstract : Avian pathogenic Escherichia coli (APEC) are known to cause extraintestinal disease in poultry, leadingto substantial losses in the industry. IutA, iron-regulated aerobactin receptor is firmly associated with APEC. To assessthe potential of IutA to induce protective immune responses, attenuated Salmonella Typhimurium strain expressingIutA was constructed and administered orally to BALB/c mice. The IutA-specific immune responses were measuredwith sera, vaginal and fecal samples by an enzyme-linked immunosorbent assay. We found that the Salmonella-IutAvaccine induced significantly higher immune responses as compared to the control inoculated with the attenuated S.Typhimurium containing the plasmid only. The IutA-specific immune responses were increased by second immunizationat third week after initial immunization, whereas triple immunization induced lower immune responses than thoseinduced by the double immunization. The Salmonella-IutA vaccine induced a nature of immunity biased to the Th1-type, as judged by the ratio of IutA-specific IgG isotypes (IgG2a/IgG1). Overall, these results suggest that theSalmonella-IutA vaccine appear to be suitable candidate for a vaccine against APEC.Keywords : avian pathogenic Escherichia coli, boost immunization, IutA, oral vaccination

Short-term and long-term antibody response by mice after immunization against Neisseria meningitidis B or diphtheria toxoid

Serogroup B Neisseria meningitidis (MenB) is a major cause of invasive disease in early childhood worldwide. The only MenB vaccine available in Brazil was produced in Cuba and has shown unsatisfactory efficacy when used to immunize millions of children in Brazil. In the present study, we compared the specific functional antibody responses evoked by the Cuban MenB vaccine with a standard vaccine against diphtheria (DTP: diphtheria, tetanus, pertussis) after primary immunization and boosting of mice. The peak of bactericidal and opsonic antibody titers to MenB and of neutralizing antibodies to diphtheria toxoid (DT) was reached after triple immunization with the MenB vaccine or DTP vaccine, respectively. However, 4 months after immunization, protective DT antibody levels were present in all DTP-vaccinated mice but in only 20% of the mice immunized against MenB. After 6 months of primary immunization, about 70% of animals still had protective neutralizing DT antibodies, but none had significant bactericidal antibodies to MenB. The booster doses of DTP or MenB vaccines produced a significant antibody recall response, suggesting that both vaccines were able to generate and maintain memory B cells during the period studied (6 months post-triple immunization). Therefore, due to the short duration of serological memory induced by the MenB vaccine (VA-MENGOC-BC® vaccine), its use should be restricted to outbreaks of meningococcal disease.

Ancylostoma ceylanicum metalloprotease 6 DNA vaccination induces partial protection against hookworm challenge infection

Hookworms are blood feeding intestinal nematodes that infect more than 500 million people and cause iron deficiency anemia. Infected children suffer from physical and cognitive growth retardation. Because of potential anthelminthic drug resistance, the need for vaccine development is urgent. Numerous antigens have been tested in animal models as vaccines against hookworm infection, but there is no effective human vaccine. We cloned a cDNA encoding Ancylostoma ceylanicum metalloprotease 6 (Acemep-6). Ace-MEP-6 is a protein with a predicted molecular mass of 101.87 kDa and based on computational analysis it is very likely to be engaged in food processing via hemoglobin digestion. Groups of hamsters were immunized with an Ace-mep-6 cDNA vaccine, either once or three times. Animals that were administered one dose developed high resistance (80%, p < 0.01) against challenge infection, whereas triple immunization resulted in no worm burden reduction. These results suggest that DNA vaccines can be powerful tools in ancylostomiasis control, although the mechanisms through which protection is conferred remain unclear.

DNA immunization with a plasmid carrying the gene of hepatitis C virus protein 5A (NS5A) induces an effective cellular immune response

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti-NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against abroad spectrum of NSSA epitopes as assessed by T-cell proliferation andsecretion of antiviral cytokines IFN-gamma and IL-2. In in vitro T-cell stimulation experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.