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Introduction. The increase in the incidence of whooping cough in children and adults of different age groups justifies the need for their revaccination and the development of new, acceptable for these purposes. This work is devoted to substantiating the design of a clinical trial and describing the results of a comparative study of the safety and tolerability of the drug "GamLPV" with two-fold intranasal administration to healthy adult volunteers using two methods. The choice of the scheme and method of administration of the drug is justified. The serological structure of the population of adults aged 18–40 years living in Moscow and the Moscow region is characterized.Aim. Determination of the safety and tolerability of the drug with a double intranasal administration of the vaccine by drip method and spraying through an actuator.Materials and methods. A randomized placebo-controlled trial included 50 healthy male and female volunteers aged 18 to 40 years who met the inclusion criteria. The volunteers were divided into 2 groups of 25 people: a drip method of administration and spraying through an "actuator". By both methods, the drug was administered twice with an interval of 60 days.Results and discussion. Serological analysis of the population of healthy volunteers at the prescreening stage justified the inclusion in the study of volunteers containing anti-pertussis antibodies in the blood (IgG ≤40 Ed/ml). A comparison of the results of preclinical studies on an experimental model of non-human monkeys and the first phase of a clinical study of GamLPV allowed us to propose two methods of double administration of the drug as a promising vaccination scheme for volunteers. A comparative randomized study shows the safety of using the proposed scheme for vaccination of adult volunteers.Conclusions. Both proposed methods of double administration can be used to plan a multicenter study to research the immunogenicity and protective activity of GamLPV.

Introduction. The Gamaleya National Center of Epidemiology and Microbiology has developed a live intranasal pertussis vaccine, GamLVP, for protection against whooping cough. It is indicated for vaccination of infants and revaccination of adults of all age groups. Preclinical studies on suckling mice or rats and adult monkeys as well as clinical trials involving adult volunteers demonstrated safety and efficacy of the GamLVP vaccine. The expansion of the GamLVP vaccine to be used for vaccination of infants requires additional preclinical studies to assess its safety and immunogenicity in the most suitable experimental model of infant hamadryas baboons (Papio hamadryas).The aim of the study was to assess safety and immunogenicity of the GamLVP vaccine administered intranasally for a single dose, two-dose, and three-dose immunization of P. hamadryas infants.Materials and methods. The study was performed in three 1–2-month-old P. hamadryas infants kept, together with their mothers, in a separate cage. The results of the complete blood count and biochemical profile tests were measured before and after the immunization and experimental infection. The enzyme immunoassay (EIA) was used to detect any changes in the levels of specific IgG antibodies in sera from the mothers and infants; the agglutination test (AT) was used to measure titers of total anti-pertussis antibodies.Results. The intranasal immunization of P. hamadryas infants with the GamLVP vaccine triggered development of a specific humoral immune response mediated by IgG antibodies (pertussis toxin + filamentous hemagglutinin), increased titers of total agglutinating anti-pertussis antibodies, caused no local and systemic reactions, caused no changes in the complete blood count and biochemical profile. The experimental infection of the GamLVPimmunized P. hamadryas infants did not cause any changes in the laboratory blood test values and any clinical manifestations typical of the pertussis infection.

Introduction. A significant increase in the incidence of pertussis in the world, including among adolescents and adults, the prevalence of mild forms of the disease and asymptomatic carrier of bacteria B. pertussis, and the resulting need for mass revaccination of different age groups determine the demand for new vaccines against B. pertussis. In N.F. Gamaleya Federal Research Center for Epidemiology and Microbiology, a live intranasal pertussis vaccine for the prevention of pertussis (GamLPV) has been developed. The GamLPV vaccine underwent preclinical studies that proved its safety and effectiveness in experiments on small laboratory animals and nonhuman monkeys. Safety of vaccine is shown in clinical studies on healthy volunteers.The aim of the study is to assess the immunogenicity of different doses of the drug GamLPV when first used in healthy volunteers.Materials and methods. The study was conducted as randomized placebo-controlled, blind trial with consistent volunteer inclusion and dose escalation. Study ID in clinicaltrials.gov database: NCT03137927 (A Phase I Clinical Study of a GamLPV, a Live Intranasal Bordetella Pertussis Vaccine). The following parameters of humoral and cellular immune responses were assessed in dynamics: levels of specific IgM, IgG and IgA antibodies in blood serum of volunteers and the number of cytokines interleukin-17, tumor necrosis factor-α, interferon-γ produced after specific induction in vitro of blood mononuclears of vaccinated volunteers. Dynamics of attenuated bacteria persistence in nasopharynx of vaccinated volunteers was evaluated.Results. Intranasal vaccination of volunteers with the drug Gam LPV resulted in the formation of a specific humoral (IgG and IgA) and cellular immune response. The dose-dependent nature of immunoglobulin and cytokine production was shown. Attenuated bacteria persisted for a long time in the nose/oropharynx of vaccinated volunteers.Discussion. Good tolerability of all tested doses of the drug justifies the choice for further investigation of a vaccine dose equal to 4 × 109 CFU. At the next stage, the safety and immunogenicity of two-time vaccination of volunteers will be studied.

Introduction. Due to the growth of laboratory-confirmed cases of pertussis among adolescents and adults, the spread of latent form of disease and the identification of asymptomatic carriage of the pathogen, the need arose to create a new vaccine against B. pertussis. On the basis of N. F. Gamaleya Federal Research Center for Epidemiology & Microbiology, a pertussis vaccine, containing attenuated B. pertussus bacteria, was developed. The article presents data on a clinical trial of the first phase of the pertussis vaccine “GumGVK, live intranasal vaccine for the prevention of pertussis” in healthy volunteers. Based on the results of the study, data shows the safety and tolerability of the first intranasal vaccine using on humans.Aim. To Study the safety and tolerability of the vaccine "GamGVK, live vaccine for intranasal use for the prevention of whooping cough»Materials and methods. Volunteers who expressed a desire to participate in the study were informed about its goals and objectives, conditions and requirements for participants, including the criteria for their inclusion/exclusion from the study. After signing the informed consent, the volunteers were screened for their compliance with the inclusion criteria. Screening studies and safety assessment of the drug were performed on the basis of anamnesis, subjective complaints, assessment of vital signs, ECG, peak flowmetry, results of laboratory tests of urine and blood, enzyme immunoassay of blood serum, analysis of nasopharyngeal aspirates for the presence of pertussis pathogen DNA, results of physical examination.Results and discussion. The study involved 36 "healthy" volunteers aged 18–40 years. The average age of the volunteers was 26.2 ± 5.5 years. Not reliably identified AES associated with the use of GumGVK. None of the used doses of the drug led to the formation of local and General allergic reactions to intranasal administration of GumGVK.Conclusions. The drug GumGVK is safe for nasal use, has good tolerance.

The aim of present research is studying of protective potency of candidate recombinant live pertussis vaccine (RLPV) for intranasal administration.Materials and methods. Two methods of protective potency assessment, based on mice survival estimating, were used: after intracerebral administration of B.pertussis 18323 bacteria and after intranasal administration of virulent Bordetella spp. bacteria (B.pertussis, B.parapertussis and B.bronchiseptica) to immunized mice.Results. An ingenious method of pertussis vaccine protective potency assessment due to mucosal immunity is suggested. Protective potency of constructed RLPV estimated in two tests of intracerebral and intranasal administration was higher than market image drug of DTP vaccine. RLPV provided protection from infection in mice after administration of both wild type and recombinant Bordetella spp. bacteria.Conclusion. Examined recombinant live pertussis vaccine (RLPV) for intranasal administration has significant protective potential and could be recommended for using in clinical trials.

Goal. Evaluation of duration and frequency of Bordetella pertussis DNA detection in contact persons in family foci of whooping-cough.Materials and methods. 116 persons from 59 family foci of pertussis were examined in contact with sick young children. The DNA of B. pertussis bacteria in nasopharyngeal swabs was detected by real-time PCR (PCR-RV) using a test system developed at Gamaleya Research Institute of Epidemiology and Microbiology (Moscow). The bacterial load and the duration of the release of genomic equivalents (GE) of B. pertussis DNA were determined in dynamics at 1, 3 and 6 months. Results. Among the contact persons in family foci, adults accounted for 59,48%, adolescents and schoolchildren – 10,35% and 12,07% respectively. Cough was absent in 35,34% of contact persons, 20,69% had a rare dry cough, 24,14% had a dry compulsive cough and 19,83% had a typical cough. None of the contact family members were diagnosed with whooping cough, although 64.66% of the patients had clinical signs of the disease, mainly its atypical form (44.83%). Among the carriers of B. pertussis adults accounted for 82.92%, among patients with atypical forms of whooping cough – 51.92%. In the study of nasopharyngeal swabs using the PCR-RV method, it was found that 86.10% of the contact persons detected DNA of B. pertussis. After 3 months in 90% of the contacts, the DNA of pertussis causative agent was detected in a minimum amount of 101-102GE/ml in the sample. After 6 months, B. pertussis was sanitized in 50% of the examined patients. 12.5% of the samples identified avirulent forms of the causative agent of pertussis, formed as a result of movement of IS481 in operon bvgAS.The conclusion. In 86.1% of contact persons in family foci for a long time (from 3 to 6 months), detection of genetic markers of the causative agent of pertussis from the nasopharynx was noted, including 35.34% of those examined in the absence of cough. This, along with the reported genetic mutation in operon bvgAS in 12.5% of cases, can characterize the presence of persistence of B. pertussis, explaining its preservation in circulation in the conditions of mass vaccine prevention.

Against the backdrop of mass parenteral immunization by present pertussis vaccines the incidence rate of pertussis is recorded in many countries, as well as among adolescents and adults, according to the World Health Organisation data. The number of cases of pertussis complicated to detect is increased, meaning atypical forms and inapparent bacteria carrying. In the presence of appearance of Bordetella pertussis strains of «new» genotype the efficacy of current pertussis vaccines is decreased. Existing epidemiologic situation cries out for development of new, more efficient pertussis vaccines and improvement of immunization methods and schedules. The most promising for this problem solution are live pertussis vaccines, constructed with genetic engineering methods.The goal of present research is studying of toxicity and safety of innovative recombinant live pertussis vaccine for intranasal administration in animals.Obtained results demonstrate the absence of specific toxicity of candidate vaccine and its use safety in intranasal applying.

Aim. The construction of recombinant attenuated Bordetella pertussis bacteria of ptxP3 genotype and its genetic and biological stability characteristics. Materials and methods. During construction of recombinant attenuated bacteria of ptxP3 genotype virulent Bordetella pertussis bacteria of ptxP1 genotype were used as a recipient. PtxP1 genotype bacteria are used for whole cell pertussis (wP) vaccine production in Russia. Mutant bacteria B. pertussis 475 were received by crossing-over between chromosome comprising native copy of target sequence and its mutant copy in recombinant suicide plasmid transferred in recipient bacteria by conjugation. Genetically engineered construction of recombinant plasmids was conducted. The structure of modified chromosome locus of attenuated bacteria was determined by PCR and amplification fragments sequence. The stability of structure and characteristics of attenuated bacteria was defined after 15 passages of strains on culture medium and 5 passages in mice. Results. Isogenic attenuated ptxP1 B. pertussis 4M and ptxP3 B. pertussis 4MKS were constructed. These bacteria produce non-toxic pertussis toxin (PT) and do not produce dermonecrotic toxin (DNT). The promoter region of ptx operon contains mutation, typical for «new» genotype of circulating virulent bacteria and increasing PT production. The structure of modified DNA fragments and characteristics of attenuated bacteria did not change while storing and after passages on culture medium and in mice. Conclusion. Recombinant attenuated bacteria B. pertussis 4MKS of «new» ptxP3 genotype are constructed. Application perspectiveness of genetically engineered modification of isogenic B.pertussis bacteria for pertussis vaccines development is shown.

The absence of an adequate experimental animal model makes difficult study of immunity against whooping cough and its pathogenesis. Experimental whooping cough reported by us earlier in pubescent non-human primates of the Old World was accompanied by specific clinical and laboratory marks in the absence of cough. The possibility of pertussis modelling while experimental whooping cough in impuberal hamadryad baboons was investigated. In the process of selection of monkeys for the further studies for perfecting of experimental model for pertussis research unexpectedly were detected specific pertussis antibodies in impuberal hamadryad baboons.The aim of the study: revealing of source of infection and transmission of pertussis to hamadryad baboons and investigation of response of antibody-positive impuberal hamadryad baboons to secondary contagion by B. pertussis bacteria while experimental infection.Results. 18 veterinary checked, somatically healthy hamadryad baboons of various gender managed in two neighboring cages. Specific pertussis IgM and IgG antibodies were found in blood serum of all the animals and one of the monkey keepers. By real-time PCR in nasopharyngeal swabs of the monkey keeper and three 7- and 9-month-old hamadryad baboons were registered single B. pertussis genom equivalents. Seropositive impuberal hamadryad baboons were experimentally challenged by virulent B. pertussis 475 strain. Quantity of B. pertussis genom equivalents and percentage of IgM and IgG antibodies in impuberal hamadryad baboons after experimental infection were detected. These results were comparable with such received after secondary experimental challenge of monkeys by B. pertussis. Humoral immuneresponse was characterized by booster effect and rapid B. pertussis elimination.Conclusion. The case of transmission of B.pertussis bacteria to hamadryad baboons by natural contagion and epizootic focus of pertussis in apery conditions were registered. In dynamics of immune response and level of bacterial load in experimentally infected impuberal and pubescent hamadryad baboons were not revealed significant differences. The possibility of asymptomatic B.pertussis transmission from man to monkey and from monkey to man without definitive spasmodic cough was reviewed. Pertussis research perspectives using experimental model of non-human primates of the Old World were marked.

A growth of pertussis morbidity is observed in many countries of the world against the background of mass vaccination. Forms of the disease course have changed. Atypical forms of pertussis occur predominately in adolescents and adults. Asymptomatic carriage of the causative agent has been established. Infection of infants with Bordetella pertussis bacteria in more than 90% of cases occurs from parents and relatives. A prolonged persistence of the causative agent has been identified. Morbidity increase in developed countries is associated with the use of acellular vaccines, that do not protect from the infection, but reduce severity of the disease. A change of genotypes of the circulating bacteria strains is observed ubiquitously. Formation of a persistent form of B. pertussis is possible due to a reversible integration of IS-elements into bvgAS operon and other virulence genes. The results of studies of invasion and survival of B. pertussis bacteria in eukaryotic cells, a change in B. pertussis bacteria population after experimental infection of laboratory mice and monkeys are presented, accumulation of avirulent insertion Bvg mutants of B. pertussis was detected. The data obtained are in accordance with the results of analysis of causative agent population in patients with typical and atypical forms of pertussis in humans. More than 50% of the population of B. pertussis bacteria in practically healthy carriers was shown to be presented by avirulent insertion Bvg mutants. B. pertussis virulence reducing as a result of inactivation of single or several virulence genes probably provide long-term persistence of bacteria in host organism and formation of apparently healthy vehicles. Follow-up studies on that front would help to formulate new attitudes to preventive measures of pertussis and lead to development of fundamentally new pharmaceuticals (vaccines) preventing formation of bacterial persistence.

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