Immunogenicity of candidate HFRS vaccines based on guinea pig models

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Introduction

Hemorrhagic fever with renal syndrome (HFRS) is an acute viral, naturally focal, zoonotic human infection that poses a serious social and medical problem due to the severity of the disease and the lack of effective etiological treatments and specific preventive measures [1, 2]. Socioeconomic losses are further exacerbated by the fact that up to 85% of HFRS cases occur in men aged 20–40, — the most active segment of the population [1, 3]. The epidemiology of human cases caused by hantaviruses is linked to the geographic distribution of a specific natural reservoir (natural host)—rodents from the families Muridae and Cricetidae [4]. Natural foci of HFRS associated with the human pathogenic hantaviruses Puumala, Kurkino, and Sochi are located in the European part of Russia, while those associated with the Hantaan, Amur and Seoul hantaviruses are found in the Far East [1].

An epidemiological analysis of HFRS incidence over the past 10 years has shown that approximately 98% of HFRS cases in Russia are etiologically caused by the Puumala hantavirus and slightly more than 2% by the Hantaan, Amur, Seoul, Kurkino and Sochi hantaviruses, indicating the leading etiological role of the Puumala hantavirus in the structure of HFRS incidence in Russia [1]. The spread of HFRS foci both in the European part of Russia and in Eastern European countries is caused by an unfavorable ecological situation and anthropogenic factors that contribute to closer contact with humans. In Asia, approximately 70% of HFRS cases are associated with the Hantaan hantavirus, while the remainder are caused by the Seoul virus [5].

Over the past 30 years, the effectiveness of inactivated HFRS vaccines has been demonstrated in China and South Korea [6]: the licensed monovalent Hantavax vaccine based on the Hantaan virus, propagated in the brain tissue of suckling mice in the Republic of Korea since 1989 [7] , and later Hantavax based on the Hantaan virus propagated in Vero cell culture [8]. In China, an inactivated beta-propiolactone vaccine based on the Hantaan and Seoul hantaviruses, is produced in gerbil kidney cells and sorbed onto aluminum hydroxide [9]. According to the literature, immunization with brain-derived vaccines based on the Hantaan or Seoul viruses is unable to prevent the development of infection caused by the Puumala virus [6]. Therefore, these vaccines cannot be used in the European part of Eurasia, as they do not provide protection against HFRS pathogens in infection foci within this territory [10]. In preclinical studies of a bivalent hantavirus vaccine based on the Puumala and Hantaan viruses, immunological cross-reactivity of virus-neutralizing antibodies with Orthohantavirus dobravaense (Dobrava, Kurkino, Sochi) was observed [10]. This allowed Orthohantavirus dobravaense to be excluded from the composition of the hantavirus vaccine. The potential of such a bivalent vaccine to cover all endemic regions of the Eurasian continent must be evaluated in clinical trials.

At the M.P. Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of the Russian Academy of Sciences (Institute of Poliomyelitis), an experimental inactivated whole-virion vaccine (EV) based on the Puumala and Hantaan viruses has been developed, which meets all requirements for immunobiological preparations administered to humans.

The aim of the study is to determine the immunogenic activity of EV against HFRS in a guinea pig model using various immunization regimens.

Materials and methods

Inactivated experimental vaccines were prepared from the Puumala and Hantaan orthohantaviruses, strains PUU-TKD/VERO and HTN-R-88/VERO, using a previously described method [11]. In summary, Puumala and Hantaan hantaviruses were propagated in Vero cell culture; pools of virus-containing culture supernatant were concentrated by tangential flow ultrafiltration, followed by the preparation of a chromatographically purified secondary concentrate with virus titers of 5.4 ± 0.2 and 5.8 ± 0.3 1og₁₀ FFU/mL, respectively, and subsequent inactivation with beta-propiolactone at a final dilution of 1:6000. After adding 0.1% human serum albumin and sterilizing filtration, the immunogenic activity of the mono- and bivalent EV based on the Puumala and Hantaan viruses was assessed.

The studies were conducted on guinea pigs (Cavia porcellus) in accordance with protocols No. 100622-5 dated June 10, 2022, No. 04072024 dated July 4, 2024, which were approved by the Ethics Committee of the M.P. Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of the Russian Academy of Sciences (Institute of Poliomyelitis). The guinea pigs were obtained from the Andreevka branch of the Scientific Center for Biomedical Technologies of the Federal Medical Biological Agency.

Before the start of the study, animals meeting the inclusion criteria for the experiment were randomly assigned to groups. The guinea pigs were kept under identical housing and feeding conditions throughout the experiment in a specialized vivarium certified for work with Group II pathogens. The animals were cared for in accordance with international and Russian regulations governing the use of laboratory animals: The European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS No. 123), Strasbourg, 1986, amended in 2006, and the “Principles of Good Laboratory Practice” (Russian National Standard GOST R 53434-2009). Female guinea pigs weighing 290–330 g were housed in accordance with GOST 33216-2014 and were randomly assigned to groups of 6 animals each according to the immunization schedule.

The EV was administered intramuscularly to the guinea pigs at a dose of 300 μL; animals in the control group received saline. In the immunization schedule, the day of the first immunization (day 0) was taken as the starting point; subsequent immunizations are designated by intervals in days relative to the first EV administration. A monovalent EV based on the Puumala hantavirus (PEV) was administered according to the following schedules: I — 0–14–364; II — 0–14–28–364; III — 0–14–50–364; IV — 0–14–182–364; V — 0–14–182–364–546; VI — 0–150–300; VII — 0–30–364; VIII — 0–30–60–364; IX — control group. For the bivalent experimental vaccine (BEV) based on the Puumala and Hantaan hantaviruses (in a 1:1 ratio), the following regimens were used: X — 0–14–182–364; XI — 0–14–50–364; XII — control group.

Blood was collected via cardiac puncture under anesthesia, and samples were processed according to the previously described method [12]. Upon completion of the experiments, all laboratory animals were euthanized. On the days of PEV and BEV administration, blood samples were collected prior to immunization. Blood samples were obtained at the time intervals specified above. The immunogenic activity of both PEV and BEV was determined by the titer of neutralizing antibodies (NAbs) in guinea pig serum in a neutralization assay (NA) based on the inhibition of focus-forming units (FFU) using the previously described method for inhibiting 50% of focus-forming units (NA/FFU₅₀) in Vero E6 cell culture [18]. Serums were warmed at +56°C for 30 min. Each blood serum sample was tested three times in the NA/FFU₅₀ assay. Data are presented as geometric mean titers (GMT) of NAb in binary logarithms. No NAbs were detected in the control groups of experimental animals (NA/FFU₅₀ cutoff limit ≤ 2.32 log₂). The presence of NAbs in the blood serum of experimental animals with a GMT ≥ 4.32 log₂ was taken as the criterion for sufficient immunogenicity [14].

The sample size for the optimal allocation of guinea pigs to the experimental groups was determined using G*Power software (ANOVA type). To achieve a statistical power of 0.8, a significance level of 0.05, and an effect size of 0.6, each experimental group included 6 guinea pigs. The results were analyzed using GraphPad Prism v. 8.4.3 software. The statistical significance of differences was determined using Sidak’s multiple comparison test.

Results

A key stage of the study was determining the magnitude and duration of the adaptive immune response generated, depending on the immunogen dosing regimen. This study identified patterns in the increase in NAb titers depending on the immunization regimen used in guinea pigs (PEV and BEV). In the blood sera of guinea pigs in both the control and experimental groups, the NAb titer was not determined at the start of the experiment. A GMT above 4.32 ± 0.2 log₂ was considered a positive NAb level [14]. nAbs were detected in animals in all experimental groups in response to the administration of PEV and BEV.

Upon administration of PEV according to regimens I–VI, a similar increase in NAb titers in blood sera was observed after the first 2 doses administered 14 days apart. Two-dose immunization induced NAb to peak values by day 42 (9.83 ± 0.35 log₂), followed by a gradual decline by day 112 (5.7 ± 0.5 log₂) from the start of immunization; NAb levels were subsequently detected until day 364 at (4.26 ± 0.22 log₂; Fig. 1, I). Booster administration of PEV at different time points led to an increase in nAb titer levels with varying intensity. A booster dose administered on day 28 after the start of immunization resulted in a statistically significant increase in NAb titer by day 56 (9.94 ± 0.4 log₂; p < 0.0001) with a rapid decline by day 96 (5.4 ± 0.36 log₂) and subsequent persistence of NAb at a level of 4.64 ± 0.39 log₂ until day 364 after the start of immunization (Fig. 1, II). A booster dose administered on day 50 induced a peak in NAbs by day 84 (12.57 ± 0.56 log₂; Fig. 1, III). Administration of PEV on day 182 induced NAb production to peak levels by day 238 (9.61 ± 0.14 log₂), which did not differ statistically significantly from those following two-dose administration. A repeated booster administration on day 364 induced NAb production to peak levels by day 462 (12.93 ± 0.13 log₂), which was statistically significantly different (p < 0.0001) from those following a single administration (Fig. 1, IV). Overall, the dynamics of the NAb titer in response to a repeat booster dose of PEV for all experimental groups was accompanied by an increase in NAb levels in guinea pig serum to peak values by days 392–406 (Fig. 1), but the most significant increase in NAb titer (12.93 ± 0.21 log₂) compared to the peak value after two-dose immunization was observed in the group of guinea pigs immunized according to the 0–14–182–364 schedule (Fig. 1, IV).

 

Fig. 1. Changes in NAb titers in guinea pig serum depending on the PEV immunization schedule: I — 0–14–364; II — 0–14–28–364; III — 0–14–50–364; IV — 0–14–182–364. A serum GMT level of ≥ 4.32 log₂ in experimental animals was taken as the criterion for sufficient immunogenicity.

 

At the same time, a trend was observed in which the duration of NAb persistence above 8 log2 increased as the time since the booster immunization increased. For a two-dose immunization regimen, NAb titers remained at their maximum level for 30 days; with a booster dose administered on day 28, they remained at that level for 40 days; on day 50, for 56 days; on day 182, for 98 days; and on day 364, for 120 days.

The observed antibody production dynamics indicated the need to immunize guinea pigs at longer intervals according to the PEV administration schedule of 0–14–182–364–546 (Fig. 2). This PEV administration regimen resulted in a statistically significant increase in NAb titer (p < 0.0001) in response to each subsequent dose, reaching maximum values (15.79 ± 0.47 log₂) by day 588. Thus, each repeated booster administration of PEV to guinea pigs induced a statistically significant increase in NAb titer compared to peak values following two-dose immunization.

 

Fig. 2. Dynamics of NAb titers in guinea pig blood sera following immunization with PEV according to the schedule V – 0–14–182–364–546. A GMT of ≥ 4.32 log₂ in the blood sera of experimental animals was taken as the criterion for sufficient immunogenicity.

 

Under the 0–150–300 immunization schedule, a single administration of PEV induced a NAb level of 5.77 ± 0.29 log₂ by day 28, followed by a decline to 4.7 ± 0.17 log₂ (Fig. 3). Administration of the second dose of PEV on day 150 induced a peak by day 182 after the start of immunization at a level of 7.66 ± 0.29 log₂, with a more gradual decline in NAb to 5.4 ± 0.51 log₂. A booster dose administered on day 300 after the start of immunization led to an increase in the NAb titer to 7.85 ± 0.37 log₂, which was not statistically significantly different from the results of two-dose immunization (Fig. 3).

 

Fig. 3. Changes in NAb titers in guinea pig blood sera following immunization with PEV according to the schedule VI — 0–150–300. A serum GMT level of ≥ 4.32 log₂ in the experimental animals was taken as the criterion for sufficient immunogenicity.

 

In immunization regimens VII–VIII, following the second dose administered 30 days after the first, a peak NAb titer of 10.0 ± 0.47 log₂ was observed by day 42 after the start of immunization, followed by a decline to 5.0 ± 0.2 log₂ by day 98 (Fig. 4). At the same time, a booster dose administered on day 364 after the start of immunization induced a lower NAb titer—at 7.86 ± 0.15 log₂ (p < 0.0001) on day 434—compared to two doses of PEV (Fig. 4, VII). With a similar PEV administration regimen but with a booster dose on day 60 after the start of immunization, a second peak was observed by day 112 (8.92 ± 0.17 log₂; p < 0.0001), which was also statistically lower than that following two doses of PEV. A repeat booster dose of PEV on day 364 induced the formation of a second peak at 7.7 ± 0.26 log₂ (p < 0.0001) by day 448, which was statistically significantly lower than the previous peak (Fig. 4, VIII).

 

Fig. 4. Changes in NAb titers in guinea pig blood sera depending on the PEV immunization schedule: VII — 0–30–364; VIII — 0–30–60–364. A serum GMT of ≥ 4.32 log₂ in the blood sera of experimental animals was taken as the criterion for sufficient immunogenicity.

 

It is worth noting that no statistically significant difference was observed in peak NAb levels following two-dose immunization of guinea pigs with a 14-day and 30-day interval (Figs. 1, 4). According to the study data, it is advisable to delay the booster immunization by one year in order to induce an adequate humoral immune response.

In the next stage of this study, we determined the immunogenic activity of the culture-inactivated BEV inactivated with beta-propiolactone. Similar NAb titers against both viruses indicate that the dose of each immunogen in the BEV was correctly selected. Immunization of guinea pigs with BEV according to the 0–14–182–364 schedule induced a similar NAb titer profile to that observed after immunization with PEV administered via a similar schedule (Fig. 5, X). Peak NAb values following two doses of the BEV were determined by day 28 at 8.47 ± 0.4 and 9.39 ± 0.41 log₂ for the Puumala and Hantaan hantaviruses, respectively, with a subsequent decline to 6. 14 ± 0.15 and 5.52 ± 0.24 log₂ by day 182. A booster dose of BEV on day 182 induced peak NAb values of 9.59 ± 0.21 and 9.59 ± 0. 11 log₂ on day 238, followed by a decrease by day 364 to 5.72 ± 0.21 and 5.3 ± 0.14 log₂. A booster dose of BEV on day 364 induced peak NAb values of 13.35 ± 0.29 log₂ on day 462 for Puumala hantavirus and 13.52 ± 0.38 log₂ on day 476 for Hantaan hantavirus, followed by a subsequent decrease.

 

Fig. 5. Changes in NAb titers in guinea pig serum depending on the BEV immunization schedule: X — 0–14–182–364; XI — 0–14–50–364. A serum GMT level of ≥ 4.32 log₂ in experimental animals was taken as the criterion for sufficient immunogenicity.

 

The maximum NAb titers in response to BEV administration according to the 0–14–50–364 schedule were observed by day 28 and amounted to 8.81 ± 0.29 log₂ for the Puumala virus, and 9.39 ± 0.4 log₂ for the Hantaan virus, followed by a statistically significant decrease by day 56 to 7.33 ± 0.15 and 7.27 ± 0.1 log₂, respectively (Fig. 5, IX). A booster dose of BEV on day 50 induced a statistically significant increase in NAb titer for the Puumala hantavirus by day 126 to 13.44 ± 0.44 log₂ and for the Hanta hantavirus — by day 140 to 13.98 ± 0.11 log₂, followed by a gradual decline by day 364 to 7.02 ± 0.16 and 6.46 ± 0.2 log₂, respectively. A booster dose of the BEV on day 364 induced NAb production to peak by day 420 (13.85 ± 0.37 and 13.1 ± 0.16 log₂ for the Puumala and Hantaan viruses, respectively).

The selected ratio of Puumala and Hantaan virus immunogens in the BEV induced a balanced humoral response. The persistence of high NAb titers in the blood sera of guinea pigs immunized with the BEV was significantly longer and lasted 220 days.

The results of this study provide the basis for selecting a vaccination schedule for clinical trials of the BEV. A balanced humoral immune response to BEV immunization allows us to consider three immunization regimens (0–150–300; 0–14–182–364; 0–30–364) as priorities for clinical studies.

Discussion

The development of vaccines against HFRS that meet current standards for medical immunobiological products administered to humans could be a decisive step toward reducing the incidence of hantavirus fevers in Russia [15]. Widespread distribution, high rates of human morbidity accompanied by a prolonged period of reduced work capacity, and the lack of specific treatments and preventive measures account for the high social and medical significance of the HFRS problem in Russia [1]. Vaccine development strategies are typically aimed at inducing both humoral (virus-neutralizing antibodies) and T-cell immunity, which generally correlates with the vaccine’s protective properties [16, 17].

The literature provides limited data on the immunogenic activity of licensed Korean and Chinese vaccines based on clinical trial results; therefore, the lower threshold of the protective level of NAbs in humans has not yet been determined. Volunteers were vaccinated with the Hantavax vaccine according to a 0–30–364 schedule. After the first dose, NAbs were detected in 13% of volunteers; after two doses, in 75%. Despite a decline in NAbs, the booster dose did not induce a significant rise in NAbs. At the same time, after one year, NAb levels had decreased to 1:10 (3.32 log₂) in 85.7% of volunteers, which corresponded to NAb levels prior to vaccination. The authors suggest that protective immunity requires frequent booster vaccinations, an alternative vaccination schedule, or changes to the vaccine formulation, such as increasing the antigen dose or adding adjuvants [7]. For the Hantavax vaccine, in Phase III clinical trials using a 0–30–364 schedule in 1,900 volunteers in the Republic of Korea, a booster effect from the third dose was observed, despite a relatively weak response to the first two doses [8].

In China, a study of the immunogenic activity of a vaccine produced in gerbil kidney cells, inactivated with beta-propiolactone, and containing aluminum as an adjuvant found that after vaccination of individuals aged 16–60 years using a 0–14–180 schedule, two doses of the vaccine induced the production of specific IgG, and against the background of their decline, a booster dose induced an increase. Thus, the study authors suggest that a three-dose immunization regimen will allow for the formation of a full-fledged immune response to the vaccine against HFRS [9].However, the study authors do not specify the protective NAb titer. However, over 30 years of vaccine use in China and Korea has contributed to a significant reduction in HFRS incidence in these countries.

However, The technological characteristics of foreign vaccines produced using Hantaan and Seoul hantaviruses, and the lack of data from preclinical studies, prevent direct comparison of their immunogenic activity with the experimental vaccine developed at the M.P. Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of the Russian Academy of Sciences (Institute of Poliomyelitis), based on the Puumala and Hantaan viruses.

In our studies, we focused on evaluating experimental vaccines based on the Puumala and Hantaan hantaviruses for the production of virus-neutralizing antibodies due to the lack of an experimental animal model that fully reproduces the clinical picture of HFRS. Previously, using a BALB/c mouse model immunized with EV on a 0–14–30 schedule, we established that after two- and three-dose immunizations, there was no statistically significant difference in either the NAb titer or the levels of interleukin-12 and interferon-γ cytokines, which allowed us to limit ourselves to a two-dose immunization regimen to assess the immunogenic activity of the experimental hantavirus vaccine preparations [15]. To investigate in greater detail how the humoral immune response depends on the immunization regimen, we selected guinea pigs as the experimental animal model, as they represent a potentially adequate due to their biological similarities to humans [18]. It was shown that the intensity of the humoral immune response is directly proportional to the dose of immunogen administered in the Puumala virus-based vaccine, and the marked increase in NAb levels following a booster dose, compared to the response after the 1st and 2nd immunizations, indicated the formation of long-lasting immunological memory [14]. The data obtained on the assessment of the immunogenicity of the mono- and bivalent experimental vaccines based on the Puumala and Hantaan viruses provide grounds for assuming high immunogenicity against both viruses in its composition for conducting clinical trials.

Conclusion

The results of this study allow for an assessment of the immunogenic activity of inactivated experimental hantavirus preparations based on the reactivity of the humoral immune response. The effectiveness of booster immunizations has been demonstrated, indicating the reliability of the immunity generated after a single administration of the hantavirus candidate vaccine. Various immunization regimens for guinea pigs have been tested, which will allow for the selection of the most effective regimen for future clinical trials in healthy adults.

About the authors

Svetlana S. Kurashova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Author for correspondence.
Email: kurashova_ss@chumakovs.su
ORCID iD: 0000-0001-9934-699X

Cand. Sci. (Med.), leading researcher, Laboratory of hemorrhagic fever

Russian Federation, Moscow

Maria S. Egorova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: egorova_ms@chumakovs.su
ORCID iD: 0000-0003-3642-6444

Cand. Sci. (Biol.), senior researcher, Laboratory of hemorrhagic fever

Russian Federation, Moscow

Anna N. Vetrova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: vetrova_an@chumakovs.su
ORCID iD: 0000-0003-1143-9732

junior researcher, Laboratory of hemorrhagic fever

Russian Federation, Moscow

Rostislav D. Teodorovich

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: teodorovich_rd@chumakovs.su
ORCID iD: 0000-0003-2117-597X

researcher, Laboratory of hemorrhagic fever

Russian Federation, Moscow

Alexandra S. Balkina

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: balkina_as@chumakovs.su
ORCID iD: 0000-0002-9704-7774

researcher, Laboratory of hemorrhagic fever

Russian Federation, Moscow

Alla V. Belyakova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: belyakova_av@chumakovs.su
ORCID iD: 0000-0003-4363-6394

Cand. Sci. (Biol.), scientific secretary

Russian Federation, Moscow

Tamara K. Dzagurova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis)

Email: dzagurova_tk@chumakovs.su
ORCID iD: 0000-0002-6656-1682

Dr. Sci. (Med.), Head, Laboratory of hemorrhagic fever

Moscow

Aydar A. Ishmukhametov

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis); Sechenov First Moscow State Medical University

Email: sue_polio@chumakovs.su
ORCID iD: 0000-0001-6130-4145

Dr. Sci. (Med.), Professor, Full Member of the Russian Academy of Sciences, General Director, Head, Department of organization and technology of production of immunobiological drugs, Institute of Translational Medicine and Biotechnology

Russian Federation, Moscow; Moscow

References

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