Multicomponent Vaccine Research Articles

Safety, Immunogenicity, and Protective Efficacy of a Chimeric A/B Live Attenuated Influenza Vaccine in a Mouse Model.

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44+CD62L−) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP366); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.

BCG, muramylpeptides, trained immunity (part II): a low molecular weight alternative to multicomponent bacterial immunostimulants for prevention of respiratory infections during a pandemic

During a pandemic, nonspecific immunoprophylaxis of SARS-CoV-2 infection and other acute respiratory infections (ARI), which can worsen the course of COVID-19, is increasingly in demand in addition to specific immunization. BCG vaccine appears to be one of the candidate immunostimulants in this regard. At the same time, other microbe-derived preparations capable of inducing a state of trained immunity deserve attention. BCG and other bacterial immunostimulatory agents containing a large number of biologically active subunits have long been considered as objects of search for promising pharmacological substances. The review analyzes the linkages between BCG, mycobacterial adjuvants, bacterial lysates, trained immunity, muramylpeptides (MPs) and NOD2 receptors in light of the choice of a low molecular weight alternative to multicomponent bacterial immunostimulants for ARI prevention during the COVID-19 pandemic. The search for key molecules by which bacteria stimulate innate and adaptive immune responses proceeds in a spiral. On different loops of this spiral, MPs have repeatedly reproduced the nonspecific effects of multicomponent bacterial adjuvants, vaccines and immunostimulants. MPs and peptidoglycans containing MPs determine the adjuvant properties of the cell walls of mycobacteria and their peptide-glycolipid fraction (wax D). MPs were able to replace Mycobacterium tuberculosis in complete Freunds adjuvant. MPs determine the NOD2-dependent ability of BCG to induce trained immunity. Probably, MPs provide NOD2-mediated long-term prophylactic action of bacterial lysates. All of the above has prompted revisiting the previously obtained evidence of the efficacy of glucosaminylmuramyl dipeptide (GMDP) as a NOD2 agonist in treatment/prevention of respiratory infections. We speculate here that MPs, in particular GMDP, at rational dosing regimens will be able to reproduce many aspects of the nonspecific effects of BCG and multicomponent bacterial immunostimulants in preventing ARI during the COVID-19 pandemic and in the post-pandemic period.

Immunogenicity of the UK group B meningococcal vaccine (4CMenB) schedule against groups B and C meningococcal strains (Sched3): outcomes of a multicentre, open-label, randomised controlled trial

The use of the multicomponent meningococcal vaccine 4CMenB in the UK schedule at 2, 4, and 12 months of age has been shown to be 59·1% effective at preventing invasive group B meningococcal disease. Here, we report the first data on the immunogenicity of this reduced-dose schedule to help to interpret this effectiveness estimate. In this multicentre, parallel-group, open-label, randomised clinical trial, infants aged up to 13 weeks due to receive their primary immunisations were recruited via child health database mailouts in Oxfordshire and via general practice surgeries in Gloucestershire and Hertfordshire. Infants were randomly assigned (1:1) with permuted block randomisation to receive a 2 + 1 (2, 4, and 12 months; group 1) or 1 + 1 (3 and 12 months; group 2) schedule of the 13-valent pneumococcal conjugate vaccine (PCV13). All infants also received 4CMenB at 2, 4, and 12 months of age, and had blood samples taken at 5 and 13 months. Participants and clinical trial staff were not masked to treatment allocation. Proportions of participants with human complement serum bactericidal antibody (hSBA) titres of at least 4 were determined for group B meningococcus (MenB) reference strains 5/99 (Neisserial Adhesin A [NadA]), NZ98/254 (porin A), and 44/76-SL (factor H binding protein [fHbp]). Geometric mean titres (GMTs) with 95% CIs were also calculated, and concomitant vaccine responses (group C meningococcus [MenC], Haemophilus influenzae b [Hib], tetanus, diphtheria, and pertussis) were compared between groups. The primary outcome was PCV13 immunogenicity, with 4CMenB immunogenicity and reactogenicity as secondary outcomes. All individuals by randomised group with a laboratory result were included in the analysis. The study is registered on the EudraCT clinical trials database, 2015-000817-32, and ClinicalTrials.gov, NCT02482636, and is complete. Between Sept 22, 2015, and Nov 1, 2017, of 376 infants screened, 213 were enrolled (106 in group 1 and 107 in group 2). 204 samples post-primary immunisation and 180 post-boost were available for analysis. The proportion of participants with hSBA of at least 4 was similar in the two study groups. For strain 5/99, all participants developed hSBA titres above 4 in both groups and at both timepoints. For strain 44/76-SL, these proportions were 95·3% (95% CI 88·5-98·7) or above post-priming (82 of 86 participants in group 1), and 92·4% (84·2-97·2) or above post-boost (73 of 79 participants in group 1). For strain NZ98/254, these proportions were 86·5% (78·0-92·6) or above post-priming (83 of 96 participants in group 2) and 88·6% (79·5-94·7) or above post-boost (70 of 79 participants in group 1). The MenC rabbit complement serum bactericidal antibody (rSBA) titre in group 1 was significantly higher than in group 2 (888·3 vs 540·4; p=0·025). There was no significant difference in geometric mean concentrations between groups 1 and 2 for diphtheria, tetanus, Hib, and pertussis post-boost. A very small number of children did not have a protective response against 44/76-SL and NZ98/254. Local and systemic reactions were similar between the two groups, apart from the 3 month timepoint when one group received an extra dose of PCV13 and recorded more systemic reactions. These data support the recent change to the licensed European schedule for 4CMenB to add an infant 2 + 1 schedule, as used in the routine UK vaccine programme with an effectiveness of 59·1%. When compared with historical data, our data do not suggest that effectiveness would be higher with a 3 + 1 schedule, however a suboptimal boost response for bactericidal antibodies against vaccine antigen fHbp suggests a need for ongoing surveillance for vaccine breakthroughs due to fHbp-matched strains. Changing from a 2 + 1 to a 1 + 1 schedule for PCV13 for the UK is unlikely to affect protection against diphtheria, tetanus, and Hib, however an unexpected reduction in bactericidal antibodies against MenC seen with the new schedule suggests that ongoing surveillance for re-emergent MenC disease is important. Bill & Melinda Gates Foundation and the National Institute for Health Research.

Synthesis and functional studies of self-adjuvanting multicomponent anti-HER2 cancer vaccines.

Breast cancer is the leading cause of cancer-related deaths among women worldwide. Human epidermal growth factor receptor 2 (HER2) overexpression is significantly associated with higher breast tumor proliferation rates. MFCH401, a 9-mer specific peptide fragment (DTILWKDIF) in the extracellular domain of the HER2 protein, is an attractive epitope for developing anti-HER2 cancer vaccines. However, the inherent low immunogenicity of MFCH401 limits its application. Herein, to induce a stronger and more durable immune response, a self-adjuvanting MFCH401-conjugated multiple-component anti-HER2 cancer vaccine was designed and synthesized by incorporating MFCH401 with lipopeptide Pam3CSK4 and a helper T cell epitope derived from tetanus toxoid P2 via an iterative condensation reaction. In vivo immunological evaluation demonstrated that the tricomponent anti-HER2 vaccine induced stronger humoral and cellular immune responses than the two-component conjugates. In addition, the induced antibodies effectively bound to HER2-overexpressing human BT474 cells. Our data clearly indicated that the MFCH401-based tricomponent anti-HER2 cancer vaccine could effectively enhance the immunogenicity of MFCH401. Structure–activity relationship analysis demonstrated that Pam3CSK4 confers better immunostimulatory activity than the helper T cell epitope P2 when conjugated with MFCH401.

Total Synthesis of a Structurally Complex Tetrasaccharide Repeating Unit of <i>Vibrio cholerae</i> O43

Vibrio cholerae is a causative agent of the deadly pandemic - cholera. Among ~200 serogroups of V. cholerae, the O1 and O139 serogroups are mainly responsible for virulent cholera. Some other serogroups are also found to be associated with diarrhoea and minor outbreaks of cholera type diseases. The deadly pathogen is a threat for many developing countries, predominately in Asia and Africa, and still causes millions of deaths every year. A multicomponent glycoconjugate vaccine covering all the strains of V. cholerae is highly desired to prevent the spread of this disease. Here, we report the first total synthesis of a densely functionalized tetrasaccharide repeating unit of V. cholerae O43, which contains rare deoxy amino sugars D-quinosamine and D-viosamine attached with a rare amino acid N-acetyl-L-allothreonine. Synthesis of orthogonally protected rare sugars and unnatural amino acid building blocks, stereoselective construction of three consecutive 1,2-cis glycosidic linkages and the presence of five nitrogen atoms dispersed over four sugar units as well as carboxylic acid functionality makes the total synthesis a formidable task. The tetrasaccharide was assembled via a highly regio- and stereoselctive [3+1] glycosylation of fully functionalized, amino acid-bearing D-viosamine donor with the trisaccharide diol. The assembly of all-1,2-cis linked trisaccharide diol was expeditiously achieved in one-pot manner. Judicious selection of protecting groups and reaction conditions enabled access to the structurally complex, amino acid-attached tetrasaccharide repeating unit, which can be used for future biological studies directed towards development of vaccine. The synthetic strategy will be useful for accessing other structurally related complex bacterial glycans.

Оценка безвредности вакцины против стрептококкозов свиней «ВЕРРЕС‑СТРЕПТО»

The production of multicomponent vaccines faces certain difficulties, since all antigens included in the composition should be of an optimal amount, not compete with each other (not cause interference) and ensure the formation of a stable and long-term immune response. However, in addition to effectiveness, it should be taken into consideration that antigen overload can have a negative effect on the safety of the drug, and, therefore, it is necessary to carefully study all possible negative reactions in naturally susceptible animals. Our work presents the results of a study of the safety of the inactivated vaccine against streptococcosis in pigs «VERRES-STREPTO» developed in the period of 2016–2020. The study was carried out on laboratory animals, as well as on piglets and sows. When using the drug according to the requirements indicated in the instructions, no local and / or systemic undesirable reactions were detected in experimental animals: anaphylaxis, depression, adynamia, fever, abortion. The formation of minor aseptic oedemas, which resolved on their own within 7–10 days, was noted at the injection site. The studies showed that the drug is safe for laboratory and naturally susceptible animals in the effective recommended and double doses. The expected efficacy of the use of this drug will significantly outweigh the potential risks of its use.

Development and evaluation of indirect enzyme-linked immunosorbent assays for the determination of immune response to multiple clostridial antigens in vaccinated captive bred southern white rhinoceros (Ceratotherium simum simum)

BackgroundAn overall increase in poaching of white rhinoceros results in captive breeding becoming a significant component of white rhinoceros conservation. However, this type of conservation comes with its own difficulties. When wildlife is captured, transported and/or confined to a boma environment, they are more predisposed to diseases caused by bacterial organisms such as spore forming Clostridium spp. A southern white rhinoceros (Ceratotherium simum simum) population on a captive bred farm was suspected to be affected by Clostridium infections. These endangered animals were apparently exposed to Clostridium spp., in the conservation area previously used for cattle farming. The rhinoceros population on the breeding operation property was vaccinated with a multi-component clostridial vaccine registered for use in cattle. Multiple indirect enzyme-linked immunosorbent assays (iELISAs) were developed in order to evaluate the serum antibody titres of these vaccinated animals. In evaluating vaccine efficacy, the gold standard mouse neutralization test (MNT) was not available and therefore iELISAs were developed for the detection of serum antibodies to C. perfringens type A (alpha toxin), C. chauvoei (whole cell), C. novyi (alpha toxin), C. septicum (alpha toxin) and C. sordellii (lethal toxin) in the white rhinoceros population using international reference sera of equine origin. Antibody titres against each clostridial antigen was evaluated in the vaccinated white rhinoceros population (n = 75). Analytical specificity showed slight cross-reactions for C. chauvoei and C. perfringens type A with the other antigens. Individual assay cut-off values were calculated with 95% confidence. Coefficient of variance (CV) values for both the international reference sera and in-house control sera across all the antigens were well below 16%, indicating good assay repeatability. This convenient and fast assay is suitable for monitoring humoral immune responses to clostridial antigens in vaccinated white rhinoceroses.ResultsCheckerboard titrations indicated optimal antigen and antibody concentrations to be used for each respective iELISA developed. Each titration set of the respective international reference and in-house control sera showed good repeatability with low standard deviations and coefficient of variance values calculated between repeats for each antigen. Individual assays proved repeatable and showed good analytical sensitivity and specificity.ConclusionsThe developed iELISAs are able to evaluate antibody profiles of phospholipase C, C. chauvoei whole cells, TcnA, ATX, TcsL in white rhinoceros serum using international reference sera.

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

4CMenB vaccine induces elite cross-protective human antibodies that compete with human factor H for binding to meningococcal fHbp.

Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal meningitis and sepsis in industrialized countries, with the highest incidence in infants and adolescents. Two recombinant protein vaccines that protect against MenB are now available (i.e. 4CMenB and MenB-fHbp). Both vaccines contain the Factor H Binding Protein (fHbp) antigen, which can bind the Human Factor H (fH), the main negative regulator of the alternative complement pathway, thus enabling bacterial survival in the blood. fHbp is present in meningococcal strains as three main variants which are immunologically distinct. Here we sought to obtain detailed information about the epitopes targeted by anti-fHbp antibodies induced by immunization with the 4CMenB multicomponent vaccine. Thirteen anti-fHbp human monoclonal antibodies (mAbs) were identified in a library of over 100 antibody fragments (Fabs) obtained from three healthy adult volunteers immunized with 4CMenB. Herein, the key cross-reactive mAbs were further characterized for antigen binding affinity, complement-mediated serum bactericidal activity (SBA) and the ability to inhibit binding of fH to live bacteria. For the first time, we identified a subset of anti-fHbp mAbs able to elicit human SBA against strains with all three variants and able to compete with human fH for fHbp binding. We present the crystal structure of fHbp v1.1 complexed with human antibody 4B3. The structure, combined with mutagenesis and binding studies, revealed the critical cross-reactive epitope. The structure also provided the molecular basis of competition for fH binding. These data suggest that the fH binding site on fHbp v1.1 can be accessible to the human immune system upon immunization, enabling elicitation of human mAbs broadly protective against MenB. The novel structural, biochemical and functional data are of great significance because the human vaccine-elicited mAbs are the first reported to inhibit the binding of fH to fHbp, and are bactericidal with human complement. Our studies provide molecular insights into the human immune response to the 4CMenB meningococcal vaccine and fuel the rationale for combined structural, immunological and functional studies when seeking deeper understanding of the mechanisms of action of human vaccines.

Multicomponent gold nano-glycoconjugate as a highly immunogenic and protective platform against Burkholderia mallei

Burkholderia mallei (Bm) is a facultative intracellular pathogen and the etiological agent of glanders, a highly infectious zoonotic disease occurring in equines and humans. The intrinsic resistance to antibiotics, lack of specific therapy, high mortality, and history as a biothreat agent, prompt the need of a safe and effective vaccine. However, the limited knowledge of protective Bm-specific antigens has hampered the development of a vaccine. Further, the use of antigen-delivery systems that enhance antigen immunogenicity and elicit robust antigen-specific immune responses has been limited and could improve vaccines against Bm. Nanovaccines, in particular gold nanoparticles (AuNPs), have been investigated as a strategy to broaden the repertoire of vaccine-mediated immunity and as a tool to produce multivalent vaccines. To synthesize a nano-glycoconjugate vaccine, six predicted highly immunogenic antigens identified by a genome-wide bio- and immuno-informatic analysis were purified and coupled to AuNPs along with lipopolysaccharide (LPS) from B. thailandensis. Mice immunized intranasally with individual AuNP-protein-LPS conjugates, showed variable degrees of protection against intranasal Bm infection, while an optimized combination formulation (containing protein antigens OmpW, OpcP, and Hemagglutinin, along with LPS) showed complete protection against lethality in a mouse model of inhalational glanders. Animals immunized with different nano-glycoconjugates showed robust antigen-specific antibody responses. Moreover, serum from animals immunized with the optimized nano-glycoconjugate formulation showed sustained antibody responses with increased serum-mediated inhibition of adherence and opsonophagocytic activity in vitro. This study provides the basis for the rational design and construction of a multicomponent vaccine platform against Bm.

Characterization of Competitive ELISA and Formulated Alhydrogel Competitive ELISA (FAcE) for Direct Quantification of Active Ingredients in GMMA-Based Vaccines.

Generalized modules for membrane antigens (GMMA) represent a technology particularly attractive for designing affordable vaccines against Gram-negative bacteria. We explored such technology for the development of O-antigen-based vaccines against Shigella and nontyphoidal Salmonella. Adsorption of GMMA on Alhydrogel was required for abrogation of pyrogenicity in rabbits, and Shigella sonnei GMMA on Alhydrogel was well tolerated and immunogenic in humans. Quantification of key antigens in formulated vaccines was fundamental for release and to check stability overtime. Traditionally, the direct quantification of antigens adsorbed on aluminum salts has been challenging, and the quantification of each active ingredient in multicomponent formulated vaccines has been even more complicated. To directly quantify each active ingredient and unbound drug substances in formulated vaccines, we developed the Formulated Alhydrogel competitive ELISA (FAcE) and the competitive ELISA method, respectively. The methods were both fully characterized, assessing specificity, repeatability, intermediate precision, and accuracy, for S. sonnei OAg quantification, both in a single component or multicomponent GMMA formulation also containing S. flexneri GMMA. The developed immunological methods allowed us to fully characterize Shigella GMMA drug products, supporting their preclinical and clinical development. The same methods, already extended to GMMA from nontyphoidal Salmonella and Neisseria meningitidis, could be potentially extended to any antigen formulated on Alhydrogel.

Quantum Chemical and Molecular Dynamics Studies of MUC1 Calix[4,8]arene Scaffold Based Anticancer Vaccine Candidates.

Functional antitumor vaccine constructs are the basis for active tumor immunotherapy, which is useful in the treatment of many types of cancers. MUC1 is one key glycoprotein for targeting and designing new strategies for multicomponent vaccines. Two self-adjuvant tetravalent vaccine candidates were prepared by clustering four or eight PDTRP MUC1 core epitope sequences on calixarene scaffolds. In this work, the different activities of two molecules with calix[4]arene and calix[8]arene skeleton are rationalized. Quantum mechanics, docking, and molecular dynamics structural optimization were first carried out followed by metadynamics to calculate the energy profiles. Further insights were obtained by complementarity studies of molecular fields. The molecular modeling results are in strong agreement with the experimental in vivo immunogenicity data. In conclusion, the overall data shows that, in the designing of anticancer vaccines, scaffold flexibility has a pivotal role in obtaining a suitable electrostatic, hydrophobic, and steric complementarity with the biological target.

Synergic complement-mediated bactericidal activity of monoclonal antibodies with distinct specificity.

The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.

Intramuscular vaccination with Strangvac is safe and induces protection against equine strangles caused by Streptococcus equi

The equine disease strangles, caused by Streptococcus equi, remains a major cause of welfare and economic cost to the global horse industry. Here we report the safety, immunogenicity and efficacy of a novel multi-component chimeric fusion protein vaccine, called Strangvac, when administered to ponies via the intramuscular route. Across the four studies, Strangvac was safe and induced robust antibody responses towards the vaccine components in blood serum and the nasopharynx, which were boosted by revaccination up to 12 months after a primary course of 2 vaccinations 4 weeks apart. The vaccine response did not cross-react with a commercial strangles iELISA, which identifies horses that have been exposed to S. equi, demonstrating that it was possible to differentiate infected from vaccinated animals (DIVA). Following challenge with S. equi strain 4047 (Se4047), all 36 control ponies that had received an adjuvant-only placebo vaccine developed clinical signs of strangles. In contrast, intramuscular vaccination with Strangvac protected ponies significantly from challenge with Se4047 at two weeks (5 of 16 ponies protected (31%), P = 0.04) and two months (7 of 12 ponies protected (58%), P = 0.0046 (including pooled control data) after second vaccination. Optimal protection (15 of 16 ponies protected (94%), P < 0.0001) was observed following challenge at two weeks post-third vaccination. Our data demonstrate that Strangvac is safe, has DIVA capability and provides a rapid onset of protective immunity against strangles. We conclude that Strangvac is a valuable tool with which to protect horses from strangles, particularly during high-risk periods, whilst maintaining the mobility of horse populations as required by the global equine industry.

Immunization with PhtD truncated fragments reduces nasopharyngeal colonization by Streptococcus pneumoniae

Despite the undeniable success of polysaccharide vaccines against Streptococcus pneumoniae infections, there is a consensus on the scientific field that this approach should be revised in order to overpass the problems related with these formulations, such as serotype replacement and high production costs. The study of conserved pneumococcal proteins or its truncated fragments has emerged as a serotype independent alternative. In this work, we have characterized the immune response elicited by systemic immunization of mice with the Histidine triad protein D (PhtD) and its’ amino and carboxyl terminal fragments. The proteins were shown to be immunogenic and protective against pneumococcal colonization, with increased IL-17 production, and induction of antibodies able to limit pneumococcal adhesion to human respiratory cells. Antiserum against PhtD_Nter, but not C_ter or PhtD, promoted an increase in bacterial phagocytosis in vitro. Interestingly, antibodies against the PhtD_Nter displayed cross-reactivity with two other pneumococcal proteins, PspA and PspC, due to sequence similarities in the proline rich region of the molecules. On a whole, our results support the inclusion of PhtD, and more specifically, its N-terminal fragment, in a multicomponent serotype independent vaccine.

Vaccination Against Tuberculosis: Revamping BCG by Molecular Genetics Guided by Immunology.

Tuberculosis (TB) remains a major health threat. Although a vaccine has been available for almost 100 years termed Bacille Calmette-Guérin (BCG), it is insufficient and better vaccines are urgently needed. This treatise describes first the basic immunology and pathology of TB with an emphasis on the role of T lymphocytes. Better understanding of the immune response to Mycobacterium tuberculosis (Mtb) serves as blueprint for rational design of TB vaccines. Then, disease epidemiology and the benefits and failures of BCG vaccination will be presented. Next, types of novel vaccine candidates are being discussed. These include: (i) antigen/adjuvant subunit vaccines; (ii) viral vectored vaccines; and (III) whole cell mycobacterial vaccines which come as live recombinant vaccines or as dead whole cell or multi-component vaccines. Subsequently, the major endpoints of clinical trials as well as administration schemes are being described. Major endpoints for clinical trials are prevention of infection (PoI), prevention of disease (PoD), and prevention of recurrence (PoR). Vaccines can be administered either pre-exposure or post-exposure with Mtb. A central part of this treatise is the description of the viable BCG-based vaccine, VPM1002, currently undergoing phase III clinical trial assessment. Finally, new approaches which could facilitate design of refined next generation TB vaccines will be discussed.

Genetic polymorphisms in malaria vaccine candidate Plasmodium falciparum reticulocyte-binding protein homologue-5 among populations in Lagos, Nigeria

BackgroundVaccines are the most reliable alternative to elicit sterile immunity against malaria but their development has been hindered by polymorphisms and strain-specificity in previously studied antigens. New vaccine candidates are therefore urgently needed. Highly conserved Plasmodium falciparum reticulocyte-binding protein homologue-5 (PfRH5) has been identified as a potential candidate for anti-disease vaccine development. PfRH5 is essential for erythrocyte invasion by merozoites and crucial for parasite survival. However, there is paucity of data on the extent of genetic variations on PfRH5 in field isolates of Plasmodium falciparum. This study described genetic polymorphisms at the high affinity binding polypeptides (HABPs) 36718, 36727, 36728 of PfRH5 in Nigerian isolates of P. falciparum. This study tested the hypothesis that only specific conserved B and T cell epitopes on PfRH5 HABPs are crucial for vaccine development.MethodsOne hundred and ninety-five microscopically confirmed P. falciparum samples collected in a prospective cross-sectional study of three different populations in Lagos, Nigeria. Genetic diversity and haplotype construct of Pfrh5 gene were determined using bi-directional sequencing approach. Tajima’s D and the ratio of nonsynonymous vs synonymous mutations were utilized to estimate the extent of balancing and directional selection in the pfrh5 gene.ResultsSequence analysis revealed three haplotypes of PfRH5 with negative Tajima’s D and dN/dS value of − 1.717 and 0.011 ± 0.020, respectively. A single nucleotide polymorphism, SNP (G → A) at position 608 was observed, which resulted in a change of the amino acid cysteine at position 203 to tyrosine. Haplotype and nucleotide diversities were 0.318 ± 0.016 and 0.0046 ± 0.0001 while inter-population genetic differentiation ranged from 0.007 to 0.037. Five polypeptide variants were identified, the most frequent being KTKYH with a frequency of 51.3%. One B-cell epitope, 151 major histocompatibility complex (MHC) class II T-cell epitopes, four intrinsically unstructured regions (IURs) and six MHC class I T-cell epitopes were observed in the study. Phylogenetic analysis of the sequences showed clustering and evidence of evolutionary relationship with 3D7, PAS-2 and FCB-2 RH5 sequences.ConclusionsThis study has revealed low level of genetic polymorphisms in PfRH5 antigen with B- and T-cell epitopes in intrinsically unstructured regions along the PfRH5 gene in Lagos, Nigeria. A broader investigation is however required in other parts of the country to support the possible inclusion of PfRH5 in a cross-protective multi-component vaccine.

A Multicomponent Vaccine Provides Immunity against Local and Systemic Infections by Group A Streptococcus across Serotypes.

Group A streptococcus (GAS) species are responsible for a broad spectrum of human diseases, ranging from superficial to invasive infections, and are associated with autoimmune disorders. There is no commercial vaccine against GAS. The clinical manifestations of GAS infection may be attributable to the large repertoire of virulence factors used selectively in different types of GAS disease. Here, we selected five molecules, highly conserved among GAS serotypes, and involved in different pathogenic mechanisms, as a multicomponent vaccine, 5CP. Intranasal (i.n.) immunization with 5CP protected mice against both mucosal and systemic GAS infection across serotypes; the protection lasted at least 6 months. Immunization of mice with 5CP constrained skin lesion development and accelerated lesion recovery. Flow cytometry and enzyme-linked immunosorbent assay analyses revealed that 5CP induced Th17 and antibody responses locally and systemically; however, the Th17 response induced by 5CP resolved more quickly than that to GAS when challenge bacteria were cleared, suggesting that 5CP is less likely to cause autoimmune responses. These findings support that immunization through the i.n. route targeting multiple nonredundant virulence factors can induce immunity against different types of GAS disease and represents an alternative strategy for GAS vaccine development, with favorable efficacy, coverage, duration, and safety.IMPORTANCE GAS is among the most common human pathogens and causes a wide variety of diseases, likely more than any other microorganism. The diverse clinical manifestations of GAS may be attributable to its large repertoire of virulence factors that are selectively and synergistically involved in streptococcal pathogenesis. To date, GAS vaccines have not been successful due to multiple serotypes and postinfection sequelae associated with autoimmunity. In this study, five conserved virulence factors that are involved in GAS pathogenesis were used as a combined vaccine. Intranasal immunization with this vaccine induced humoral and cellular immune responses across GAS serotypes and protected against mucosal, systemic, and skin infections. The significance of this work is to demonstrate that the efficacy of GAS vaccines can be achieved by including multiple nonredundant critical virulence factors and inducing local and systemic immunity. The strategy also provides valuable insights for vaccine development against other pathogens.

Dissolving undercut microneedle arrays for multicomponent cutaneous vaccination

The skin is an attractive tissue target for vaccination, as it is readily accessible and contains a dense population of antigen-presenting and immune-accessory cells. Microneedle arrays (MNAs) are emerging as an effective tool for in situ engineering of the cutaneous microenvironment to enable diverse immunization strategies. Here, we present novel dissolving undercut MNAs and demonstrate their application for effective multicomponent cutaneous vaccination. The MNAs are composed of micron-scale needles featuring pyramidal heads supported by undercut stem regions with filleted bases to ensure successful skin penetration and retention during application. Prior efforts to fabricate dissolving undercut microstructures were limited and required complex and lengthy processing and assembly steps. In the current study, we strategically combine three-dimensional (3D) laser lithography, an emerging micro-additive manufacturing method with unique geometric capabilities and nanoscale resolution, and micromolding with favorable materials. This approach enables reproducible production of dissolving MNAs with undercut microneedles that can be tip-loaded with multiple biocargos, such as antigen (ovalbumin) and adjuvant (Poly(I:C)). The resulting MNAs fulfill the geometric (sharp tips and smooth edges) and mechanical-strength requirements for failure-free penetration of human and murine skin to simultaneously deliver multicomponent (antigen plus adjuvant) vaccines to the same cutaneous microenvironment. Cutaneous vaccination of mice using these MNAs induces more potent antigen-specific cellular and humoral immune responses than those elicited by traditional intramuscular injection. Together, the unique geometric features of these undercut MNAs and the associated manufacturing strategy, which is compatible with diverse drugs and biologics, could enable a broad range of non-cutaneous and cutaneous drug delivery applications, including multicomponent vaccination.

The Problem of Timeliness of Young Children Vaccination and Ways to Solve It

Relevance Aims . Despite of widespread achievements and maintenance of appropriate levels of coverage of preventive vaccinations (&gt;95%) the problem of delayed immunizations and vaccination on time is still existed in the Russian Federation over the last years. Materials and methods . A selective analysis of the vaccination on time against diphtheria, tetanus, pertussis and hepatitis B, of children aged under 2 years in Russia, Moscow, from 2014 to 2018 was conducted. To assess the timeliness of vaccination, two independent sample studies were conducted: the vaccination history of 6003 whooping cough patients in Moscow was analyzed and vaccination was evaluated in 641 children, who were born 2016–2018 in Kirov, depending on the implementation of combination vaccines. Results . The current 95% coverage of preventive age-recommended vaccination of decreed age against pertussis, diphtheria, tetanus, polio, hepatitis B is achieved only by 2–3 years of age. Conducted analysis showed, that the implementation of combination vaccines contributed to increase in the proportion of timely vaccinated children up to 1 year of age as well as propotion of children who started primary immunization series at recommended age of 3 months. Also as a result the vaccination coverage against Hib infection and proportion of timely vaccinated against pneumococcal infection was increased. Conclusions . The implementation of modern multicomponent combination vaccines allows to increase vaccination coverage in population and to control effectively infectious diseases and also to reduce number of injections and visits to clinics.