Reactogenicity, safety and immunological efficacy of the live, pentavalent rotavirus vaccine in childhood immunization (results of the multicenter clinical trial)

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Introduction

Rotavirus infection (RVI) is the most widespread enteric infection almost in all countries of the world. According to data from WHO experts, among 1.0-1.2 billion diarrheal disease cases reported annually, viral infections account for 49-67% [1][2]. The factors con­tributing to the high prevalence of RVI are as follows: Impossibility to fight effectively the source of infection due to a large number of carriers and lack of etiotro- pic therapy, uncontrollable modes of transmission, high contagiousness of the virus and low efficiency of disin­fectants [3][4][5]. Being highly contagious (children can excrete >10 billion viral particles per milliliter of feces, while the minimal infective dose is 10 plaque-forming units/ml), RVI is one of the most common causes of hospital-associated diarrhea cases, which during some seasons can reach 87% [6][7][9].

According to the data from WHO experts, the RVI incidence varied widely across countries: 250-3,000 cases per 100,000 children. Every year, the Unites States reports more than 1 million cases of severe ro­tavirus-related diarrhea among patients aged 1-4 years [10][11][12][13]. The US-based large-scale studies showed that before the RVI vaccination program was launched, 80% of children had been seropositive, i.e. had already experienced rotavirus infection. The similar studies conducted in Poland in 2008 confirmed RVI presence almost in every third child with acute enteric infection [14][15].

According to WHO, RVI is the leading cause of gastroenteritis in children younger than 5 years of age both in high-income and low-income countries. A sig­nificant difference is observed only in mortality rates being much higher in developing countries, which ac­count for 82% of all RVI deaths worldwide.

Yet, RVI remains to be a pressing problem in de­veloped countries. European experts have estimated that there are 3.6 million episodes of rotavirus gas­troenteritis annually among 23.6 million children under the age of 5, living in European Union coun­tries; the estimated annual rate of clinical rotavirus gastroenteritis is 1 case in every 7 children [16]. The infection is responsible for 231 deaths, more than 87 thousand hospitalizations, and nearly 700 thousand outpatient visits annually. In EU countries, the rotavi­rus-associated mortality is low; however, it is RVI that contributes to high incidence among pediatric popu­lation demonstrating a large number of clinical forms requiring hospitalization and emergency room visits. The average length of a hospital stay is 2-10 days; the cost of hospital stay is 1,500 euros [15]. In the total number of cases of acute gastroenteritis in chil­dren under 5 in European countries, the proportion of rotavirus gastroenteritis ranges from 25.3% (Greece) to 63.5% (Norway). In other countries, this proportion is 36-45% [16][17].

According to the WHO data, RVI holds the second place after pneumococcal disease in the child mortali­ty caused by vaccine-preventable diseases [18][19][20][21]. In 2011, diarrheal disease accounted for 9.9% of 6.9 mil­lion deaths in this age group, where more than 70% of the deceased were younger than 2 years [14][18][19]. Among the etiologically deciphered viral diarrheas, RVI is detected in 65% of cases, being responsible for 17.8% fatal diarrhea cases. Thus, 197 thousand deaths are annually attributable to rotavirus gastroenteritis, meaning that 23 children die of the infection every hour [14][18][20][21].

In Russia, among acute enteric infections, the virus infections (rotavirus and norovirus infections) account for more than 50%. In Russia, RVI incidence has been demonstrating upward trend for the last 10 years, and in 2018 it was responsible for 81.3 cases per 100 thousand people. Apparently, this dynamics can be attributed to improved quality of laboratory diagnostics of acute en­teric infections, thus giving a better picture of the main viruses causing acute gastroenteritis in Russian popula­tion [22]. The virus carriage rate among young children is 1.5-9.0% [23].

Preventive vaccination is the most effective mea­sure against this infection. At present, vaccination against RVI is adopted in 69 countries and is includ­ed in immunization schedules in the United States, Belgium, Germany, Austria, some countries of Latin America, etc. [24][25][26]. The vaccination series con­sists of 2-3 doses administered orally at 4 to 8-week intervals during the first 6 months of life (concomi­tantly with diphtheria, tetanus, pertussis, polio and haemophilus influenza type B vaccines). The immu­nization effectiveness reaches 70-80% protection and shows 100% reduction in hospitalization or in­travenous rehydration cases [27][28]. In Mexico, RVI vaccination during the first 3 years resulted in 700 fewer deaths a year among children from the time of vaccination [25]. The Russian National Immuniza­tion Calendar (NIC) does not include immunization of children against RVI. Vaccination is performed only in some regions of Russia in accordance with regional immunization programs (schedules).

Two attenuated rotavirus vaccines are currently li­censed and successfully used worldwide — pentavalent reassortant RotaTeq (Merck Sharp & Dohme B.V., The Netherlands) and monovalent Rotarix (GlaxoSmith-Kline Trading, Belgium). Only one vaccine, RotaTeq, has been registered in the Russian Federation. It con­tains 5 reassortant rotaviruses: 4 rotaviruses express VP7 outer capsid proteins of serotypes G1, G2, G3, G4 from the human rotavirus parent strain and the VP4 protein of serotype P7 from the bovine rotavirus par­ent strain. The fifth reassortant virus expresses the P1A protein from the human strain and the G6 protein from the bovine rotavirus parent strain [29][30]. Russia has no home-produced vaccine against RVI.

In 2014, a new thermostable oral pentavalent vac­cine based on bovine rotaviruses was created in India. Since 2018 it has been internationally available as a WHO-prequalified vaccine. This immunological for­mulation was tested in India where it demonstrated high efficacy (66.7%) [30][31].

The clinical trials (phases I, II, and III) of the live, pentavalent RVI vaccine (BRV-PV) (Serum Institute of India Ltd., India) were conducted in India and Niger, with more than 15 thousand people scheduled for par­ticipation. The studied groups included adults, young children and infants. Safety and good tolerability of BRV-PV was demonstrated by all age groups during the clinical trials. No serious unsolicited events were re­ported. A few reported unsolicited events were mild to moderate and transient. No virus shedding was detected in stool samples.

The conducted clinical trials demonstrated the highest immunogenicity and protective efficacy of BRV- PV when administered to children aged 4-6 weeks as a 3-dose series. Three doses of the vaccine were adminis­tered concomitantly with diphtheria, tetanus, pertussis, haemophilus influenza type B, hepatitis B, and polio vaccines. This approach helped significantly expand the BRV-PV immunization coverage and cut expenses incurred by parents when visiting vaccination centers.

Considering the foregoing and to have the vaccine officially registered in Russia, healthy volunteers aged 18-45 years were invited to participate in a prospective, randomized, doubleblind, placebo-controlled clinical trial of safety and efficacy of BRV-PV. The trial was conducted in compliance with the Clinical Trial Proto­col No. РТВ 001/18. The Ministry of Health ofthe Rus­sian Federation issued the authorization for conducting clinical trials in children (No. 46, 29/1/2019).

The purpose of this study is to assess reactogenicity, safety and immunological efficacy of BRV-PV with participation of children. It is our first study of this vaccine during immunization of young children in Russia.

Materials and methods

Reactogenicity, safety and immunological effica­cy of BRV-PV were studied during the multicenter pro­spective, randomized, double-blind, placebo-controlled clinical trial with participation of healthy children 2 months of age at the time of the first vaccination.

Bovine and human reassortant strains of serotype G1, G2, G3, G4 and G9 were received by the Serum In­stitute of India Ltd. (India) from Dr. Kapikian (The Na­tional Institutes of Health, USA). The Serum Institute of India Ltd. prepared the primary virus strain vaccine bank, the secondary virus strain vaccine bank and the working virus strain vaccine bank to be used in manu­facturing of monovalent bulk vaccine. During manu­facturing of future ready-to-use formulations, sucrose and glycine were used as stabilizers to provide stability of the vaccine at different temperatures.

The clinical trial with participation of children was conducted in compliance with the ethical rules and reg­ulations specified in the Declaration of Helsinki (2013) and practice guidelines applicable to clinical trials con­ducted in Russia.

The trial was conducted in two clinical centers: Perm and Tyumen.

Inclusion criteria: healthy infant males or females aged 2 months at the time of the first vaccination, hav­ing no contraindications to immunization, whose ges­tational age is >37 weeks, birthweight is >2,500 g and who are vaccinated in accordance with their age-related immunization schedule. To have their child enrolled, the parents must sign their informed consent by their own hand and voluntarily as well as adhere to the re­quirements of the protocol.

Exclusion criteria:

• pre-existing diarrhea or blood in stool history or defecation disorder during the last 14 days;
• chronic diseases of the gastrointestinal tract, intussusception and congenital gastrointestinal tract defects;
• abdominal surgery;
• allergy to any components of the vaccine or to any previous vaccination;
• severe vaccine-induced reactions and complica­tions associated with any previous vaccination;
• existence of any systemic disorder in lungs, the liver, kidneys, skin, the cardiovascular system, the gastrointestinal tract, the endocrine system, the immune system, the nervous system as well as oncology or autoimmune disease;
• congenital or genetic disorders;
• acute infectious or non-infectious diseases at the time of enrollment or less than 4 weeks after re­covery;
• history of confirmed hepatitis B, diphtheria, tet­anus, pertussis, polio, haemophilus influenza or pneumococcal disease;
• confirmed or suspected immunodeficiency condi­tion (inherited or congenital immunodeficiency);
• continuous use of immunosupressors or immu- nomodulators, steroids, immunoglobulins or blood components.

The trial consisted of 3 phases. Phase I included a screening period for max 7 days and a 30-day follow-up period of primary monitoring of the child’s develop­ment after the 1^st dose of BRV-PV.

Phase II included the 2^nd dose of BRV-PV and a 45-day follow-up period of secondary monitoring of the child’s development.

Phase III included the 3^rd dose of BRV-PV and a 30-day follow-up tertiary monitoring of the child’s de­velopment in accordance with the universally accepted pediatric standards. Based on the data of clinical and laboratory studies a total of 100 children with the veri­fied "good health" status were enrolled in the trial. All of them met the inclusion criteria and did not have any condition qualifying them for exclusion.

All the children were randomized to one of the two groups at a 1:1 ratio: The 1^st group (n = 50) of children received three doses of BRV-PV orally at an interval of min 4 weeks, a 2.5 ml dose; the 2^nd group (n = 50) of children received three placebo doses at an interval of min 4 weeks, a 2.5 ml dose.

All the children received other preventive vac­cines in accordance with NIC: against pneumococcal disease (the first vaccination at the age of 2 months; the second vaccination at the age of 4.5 months), diph­theria, pertussis, tetanus, polio (the first vaccination at the age of 3 months; the second vaccination at the age of 4.5 months). The above vaccines could be received concomitantly with BRV-PV/placebo.

The reactogenicity and safety of BRV-PV were assessed by using physical examination, anthropome­try, heart rate, respiratory rate, and body temperature measurements, frequency of occurrence of postvaccinal reactions, unsolicited events and laboratory tests during active (outpatient visits and phone calls) and passive (post-immunization diary cards filled out by the par­ents) monitoring of the trial participants.

After each dose of BRV-PV or placebo, all the participants were observed at the research center for 2 hours. Thirty and 60 minutes after the BRV-PV/placebo intake, the physician-researcher examined the children physically and measured the main vitals. Six hours af­ter each vaccination, the physician-researcher made a phone call to the child’s parents.

The postvaccinal reaction was assessed with a 4-point score and the following criteria:

• the reaction is not intense;
• mild reaction —slight symptoms, hyperthermia of 37.0-37.5°C;
• moderately intense reaction — symptoms no­ticeably interfering with the normal daily activi­ties, hyperthermia of 37.6-38.5°C;
• severe reaction — symptoms impeding the nor­mal daily activities, hyperthermia >38.6°C.

The laboratory tests included complete blood count (the hemoglobin level, erythrocyte sedimentation rate, formed elements, white blood cell count, relative plate­let count, red blood cell count) and blood biochemistry (levels of alanine and aspartate aminotransferase, γ-glu- tamyl transferase, total bilirubin, total protein, blood urea nitrogen, creatinine, C-reactive protein, and glucose), a urinalysis (amounts of albumin, glucose, cell elements, salts, urine specific gravity, pH) and measurement of the total IgE level in blood sera of the participants.

The immunological efficacy of BRV-PV was assessed by rotavirus antibody (IgA) titer levels in the children’s blood sera before and after three-dose BRV-PV vaccination. The anti-rotavirus IgA was ex­amined at the Wellcome Trust Research Laboratory of the Christian Medical College (India) by using an en­zyme-linked immunosorbent assay (ELISA) in accor­dance with the approved standard operating procedure, WTRL/ASA/035 "Detection of anti-rotavirus IgA with ELISA assay and 1% reagent for western blotting." The validated analytical procedure of the ELISA assay is designed for performing quantitative measurement of rotavirus IgA antibodies in human blood serum sam­ples when analyzing results of clinical trials of rotavi­rus vaccines.

The assay was performed on purified rabbit anti-rotavirus IgG antibodies as immobilized antibod­ies for conjugation of rotavirus lysates. The conjugated rotavirus lysate immobilizes the anti-rotavirus antibody present in the test sample. The anti-rotavirus IgA anti­body is detected by the biotinylated IgA secondary anti­bodies. The detection is amplified by using the prepared avidin-biotin horseradish peroxidase complex. The concentration of the rotavirus-specific IgA in the test sample was derived by extrapolation from the standard curve generated from several dilutions of the reference serum with the assigned quantity of anti-rotavirus IgA (in U/ml).

The laboratory tests were performed on 197 blood serum samples from young children who took part in the trial. The samples were stored at -20°C prior to the tests.

The immunogenicity of BRV-PV was assessed by the following parameters:

• geometric mean concentration (GMC) of IgA antibodies in children before the vaccination in min 4 weeks after the 3^rd dose of the vaccine);
• seroconversion rate — the number and percent­age of the children with a 2, 3, 4-fold and grea­ter increase in IgA antibody titer in min 4 weeks after the 3^rd dose of the vaccine as compared to the initial level;
• seroconversion factor — the ratio between GMC of IgA antibody titers in min 4 weeks after the 3^rd dose of the vaccine and the initial titer level.

Results

During the trial, there were 55 postvaccinal reac­tions recorded in 25 participants.

No postvaccinal reactions were recorded 60 min­utes after the 1^st, 2^nd and 3^rd doses of the vaccine. Based on the phone calls made 6 hours after the 1^st immuni­zation, there were 7 postvaccinal reactions reported by the parents of 2 participants from the 1^st group, namely irritability, restlessness, bloating, decreased appetite and elevated body temperature to 37.6°C. No postvac­cinal reactions were reported in the 2^nd group within 6 hours.

During 7 days after the 1^st dose of the vaccine, a total of 11 postvaccinal reactions were recorded in 4 participants from the 1^st group and 13 postvaccinal reactions were recorded in 5 participants from the 2^nd group. Further on, during 7 days, 10 reactions were recorded in the vaccinated group after the 2^nd dose of the vaccine, and 1 reaction was recorded after the 3^rd dose. At the same time, 1 and 7 reactions were record­ed, respectively, in the placebo-controlled group. Inten­sity-based characteristics of the postvaccinal reactions during all the trial phases are given in Table 1.

 

 

Thus, during the 7-day monitoring after the three- dose series vaccination, a total of 43 postvaccinal re­actions were recorded. They were mild or moderate, except for one reaction accompanied by high fever in a child from the 2^nd group. The corrective treatment aimed at removal of unsolicited symptoms was used only in 2 cases: To reverse the fever response after the 3^rd dose in a child from the 2^nd group and to reverse such symptoms as bloating and bellyaches after the 1^st dose in a child from the 2^nd group. All the reactions were short-term (lasting from several hours to 3 days) and were resolved in recovery without consequences.

No statistically significant differences in the fre­quency of the recorded postvaccinal reactions were de­tected between the groups.

Note that bellyaches, fatigue and crying were ob­served only in children from the 2^nd group, thus excluding any connection with the BRV-PV administration. Furthermore, such reactions as elevated body tempera­ture, irritability, crying, decreased appetite, and diar­rhea are typical of Prevnar® 13 and Pentaxim® vaccines administered to the trial participants concomitantly with the studied vaccine in accordance with NIC. The postvaccinal reactions recorded during the 7-day mon­itoring period after the BRV-PV administration may have been caused by the concomitant vaccination.

During the entire monitoring period, along with the postvaccinal reactions, the children had 12 adverse events following the immunization and displayed as adverse symptoms, complaints or intercurrent diseases unrelated to the immunization, out of which 9 cases were recorded in children from the 1^st group and 3 cas­es were observed in children from the 2^nd group. All of them were characterized by mild to moderate severity, except for the elevated body temperature to 38.7°C in 1 child from the 2^nd group, making it the case charac­terized as a severe condition. No statistically significant differences in the frequency of occurrence and sever­ity level of adverse events were revealed between the groups after the immunization.

The obtained results for BRV-PV safety are com­patible with the results obtained during the clinical trials of the RotaTeq vaccine, which is registered and successfully used in Russia. During the trials aimed to study the safety of the RotaTeq vaccine, unsolicited re­actions were recorded during 42 days after the vaccina­tion. The most frequently occurring unsolicited events were upper respiration tract infections, diarrhea, vomit­ing, acute otitis media, cough and irritability [29].

Based on the data from the foreign studies, the in­take of the rotavirus vaccine was frequently associat­ed with an increased incidence rate of intussusception, a rare form of bowel obstruction [32][33]. During our clinical trial, no postvaccinal complications, including intussusception, as well as adverse unsolicited events were recorded.

The analysis of the impact of the vaccine on the complete blood count, blood chemistry and urinalysis results showed that all the laboratory test values record­ed throughout the monitoring process were within the physiological range; no significant deviation from the baseline values was detected.

No allergenic characteristics were found in BRV- PV — the serum IgE levels did not demonstrate signifi­cant changes during the monitoring period.

No negative changes in the health condition of the children vaccinated both with BRV-PV and placebo were found. The children developed harmoniously, ac­cording to their age.

The results of the conducted laboratory tests con­firmed that BRV-PV vaccination did not have any ad­verse impact on the main values of the complete blood count, blood biochemistry and urinalysis.

Thus, the obtained results were indicative of the satisfactory safety profile of BRV-PV when adminis­tered as a three-dose series childhood immunization. The obtained results were compatible with the data from foreign and Russian authors [30][32].

The assessment of the immunological efficacy of BRV-PV showed that the children - participants of the clinical trial - who were seropositive and had rotavi­rus-specific IgA concentration > 20 U/ml at the time of their enrollment in the trial, prior to the 1^st dose account­ed for 20.8% in the vaccinated group and 32.7% in the placebo-controlled group, thus confirming the previous data on infants’ seropositivity that can be associated with high levels of the impact of natural rotavirus in­fection at a very young age (previously obtained results of the clinical trials of the studied vaccine in India and Niger), placental transmission of maternal antibodies to the child as well as the alimentary mode of transmission of maternal antibodies through breast milk [34].

The three-dose series vaccination of seropositive children made it possible to reach the 50% seroconver­sion rate. In the group of initially seronegative partici­pants, 92.1% of the BRV-PV vaccinated had a fourfold and greater increase in the specific antibody titer. The increase in the level of specific IgA antibodies after the three-dose series vaccination of BRV-PV in the 1^st group was significantly different from the increase in the IgA level in the placebo group (t = 2.020; p = 0.047).

The results of the assessment of the immunolog­ical efficacy of BRV-PV in the group of seronegative and seropositive children - participants of the trial - are given in Table 2.

 

Discussion

The obtained results demonstrate excellent im­munogenic properties of BRV-PV and prove that with three-dose immunization, seroconversion can be ob­served even in the individuals who were initially se­ropositive, thus confirming the previously obtained re­sults of clinical trials. It can also imply high potential efficacy of the vaccine [35].

Thus, the conducted trials lead to the conclusion that BRV-PV is characterized by a satisfactory safety profile and high immunogenicity when administered as a three-dose series immunization to children. It can be recommended for registration as a vaccine for immuni­zation of infants from 2 months of age. The solution on localization of RVI vaccine production at Russian facto­ries, provided that the two registered vaccines are com­patible in terms of their safety and immunogenicity, can be based on the results of a pharmacoeconomic analysis.

About the authors

Irina V. Feldblium

Perm State Medical University named after Academician E.A. Wagner

Author for correspondence.
Email: irinablum@mail.ru
ORCID iD: 0000-0003-4398-5703
Dr. Sci. (Med.), Prof., Head, Department of epidemiology and hygiene Russian Federation

Ksenya A. Subbotina

Perm State Medical University named after Academician E.A. Wagner

Email: ka.subbotina@bk.ru
ORCID iD: 0000-0002-0060-6251
PhD (Med.), Assoc. Prof., Department of epidemiology and hygiene Russian Federation

Olga A. Rychkova

Tyumen State Medical University

Email: fake@neicon.ru
ORCID iD: 0000-0002-9468-6562
Dr. Sci. (Med), Head, Department of children's diseases, Faculty of medicine with a course of immunology and allergology Russian Federation

Aleksander N. Mironov

National Agency of Medicines Ltd.

Email: fake@neicon.ru
ORCID iD: 0000-0003-2969-2402
Dr. Sci. (Med.), Medical director Russian Federation

Daria A. Volkova

Perm State Medical University named after Academician E.A. Wagner

Email: fake@neicon.ru
ORCID iD: 0000-0002-7388-9950
graduate student, Department of epidemiology and hygiene Russian Federation

Azamat O. Metov

PHARM AID LTD

Email: fake@neicon.ru
ORCID iD: 0000-0003-0794-0524
general director Russian Federation

Irina V. Sakaeva

National Agency of Medicines Ltd.

Email: fake@neicon.ru
ORCID iD: 0000-0002-4662-4105
deputy general director Russian Federation

Natalia V. Kupina

CTR-Pharma Ltd.

Email: fake@neicon.ru
ORCID iD: 0000-0001-5696-9919
PhD (Med.), general director Russian Federation

Mikhail S. Karbyshev

PHARM AID LTD

Email: fake@neicon.ru
ORCID iD: 0000-0001-5969-3874
Head, Technology transfer and scientific research Russian Federation

Nikita A. Nikita A. Mironov —

National Agency of Medicines Ltd.

Email: fake@neicon.ru
ORCID iD: 0000-0001-6729-4371
project manager of preclinic and clinic researches, Department of preclinic and clinic researches Russian Federation

Yaroslava Yu. Kondratyeva

CTR-Pharma Ltd.

Email: fake@neicon.ru
ORCID iD: 0000-0001-8943-5374
quality director Russian Federation

References

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