Introduction

The SARS-CoV-2 pandemic, which has affected over 200 million people and killed 4.3 million people globally as of August 2021, has been difficult to control because of its heavy economic, healthcare, and social costs [1]. Thus, the pandemic has created an urgent need for rapid vaccine development based on scientific discoveries and our understanding of the virus biology, variation, and transmission potential [2]. Despite the time required to develop safe and effective vaccines (typically 1–2 years), vaccination remains the most effective and cost-efficient strategy for achieving “herd immunity” [3]. According to Anderson et al., vaccination of up to 90% of the population may be necessary to achieve COVID-19 herd immunity [4]. While each COVID-19 vaccine has its unique benefits and drawbacks, these vaccines must be widely accessible and affordable, especially in developing nations, to contain the pandemic [5].

The human body’s initial response to infection by the SARSCoV-2 virus is to secrete cytokine IL-6, which is believed to modulate both innate and adaptive immunity. The virus causes a capillary leak, endothelial cell destruction, and macrophage activation. The continuous generation of IL-6 leads to cytokine storm, ultimately resulting in the clinical and pathological features of acute respiratory distress syndrome (ARDS) [6]. SARS-CoV-2 also triggers an immune response, including B cell activation to produce antibodies against specific antigens. Neutralizing antibodies, the most common strategy for treating viral infections, should be used to minimize SARS-CoV-2 transmission. This strategy requires vaccination to strengthen immune defenses [7].

A successful vaccine should induce a strong Th1 and a weak Th2 response. This is because a robust Th1 cell response can trigger protective humoral and cell-mediated immunity and reduce the risk of disease enhancement, while excessive Th2 activity can cause uncontrolled tissue damage and negate the Th1-mediated microbicidal effect [8]. The Th1/Th2 balance in COVID-19 is a prognostic indicator of the disease outcome, with 78% of patients who died having an overactive Th2 response. This suggests that the type of Th response is essential for disease resolution. A wellregulated Th1-mediated immune response can effectively clear the virus, but a poorly organized response can lead to an exacerbated Th2-mediated cytokine storm, which is strongly associated with poor prognosis. Ideally, for optimal protection against SARSCoV-2 exposure, high neutralizing antibodies titer, a robust cytotoxic CD8+ T cell response, and a Th1-biased CD4+ effector response are essential [9].

Live vaccinations may pose a risk to individuals with immunosuppression or weakened immune systems. Inactivated viruses may be safer, but they often provide less long-lacting protection and require repeated booster doses [10]. Inactivated viral vaccines are both rapid and reliable to raise protective immunity, and have been widely used to prevent emerging infectious diseases. However, caution is warranted in developing COVID-19 vaccines, as SARS-CoV-2 infection can exhibit antibody-dependent enhancement, highlighting the importance of safety assessment.

In a previous study, we described the development of the inactivated SARS-CoV-2 vaccine candidate EGYVAX, including sample collection, virus isolation, propagation, virus inactivation, and vaccine production, as well as safety and immunogenicity evaluation of two doses of the vaccine in mice [11]. Here, we report the results of the preclinical dose-finding studies of EGYVAX, demonstrating its safety and immunogenicity at different vaccine doses in rhesus macaques. This paves the way to initiate the firstin-human clinical trial for a SARS-CoV-2 vaccine candidate in Egypt.

Material and Methods

The preclinical studies of the inactivated EGYVAX vaccine were conducted in accordance with the Food and Drug Administration (FDA) guidelines for developing and licensing SARS-CoV-2 vaccines [12], which are based on the World Health Organization (WHO) guidelines for the preclinical development of vaccines [13] including the required study designs, number of doses, related investigations, and procedures.

Experimental animals

Ten rhesus macaque monkeys with an equal ratio of males and females, aged 7–14 years and weighing 9–16 kg, were obtained from Giza and Alex Zoo. The animals were acclimatized for 7 days before dosing. The monkeys were housed in standard macaque cages at a temperature of 25°C (±3°C), relative humidity of 30% to 70%, and a 12-hour dark-light cycle. They were fed a standard macaque diet and given tap water at the Egyptian Army Veterinary Hospital from March 23, 2021, to May 04, 2021. Animals were identified by their cage number for the duration of the experiment. The observation period was 42 days. We used a sample size of 10 animals based on availability and literature precedent [14].

Six experimental macaque monkeys (n = 6) were randomly assigned to receive three doses (0.5 ml) of inactivated vaccine containing 35, 70, and 140 µg total protein, administered by intramuscular (IM) injection on days 0, 14, and 28 (initial and two booster doses considering the N+1 rule). Four control animals were injected with 0.5 ml saline at the same time points. Two control animals 70 and 140 µg protein of, the inactivated virus, EGYVAX, while previously immunized with the vaccine 7 months apart were used group 4 which served as negative control Animals were randomly allocated into five groups (n=10; 5 males & 5 females) each of 1 male and 1 female. Groups 1-3 received 35, 70 and 140 µg protein of, the inactivated virus, EGYVAX, while group 4 which served as negative control received saline IM doses and group 5 which served as positive control were previously immunized with EGYVAX and received saline IM doses.

Table 1: Dosing scheme of the study.

Animals were assessed daily for both clinical signs and mortality. Cellular, biochemical, and molecular analyses were conducted, including complete blood count (CBC), serum chemistry, serum neutralizing antibody (NAB) titers, immunophenotyping, chest X-ray, and RT-PCR for detecting viral RNA.

1-  General clinical signs & Mortality:

Morbidity and mortality were monitored twice daily. Clinical observations were performed using a general welfare assessment score sheet adapted from Wolfensohn & Honess, 2005 [15]. Animal appearance and behaviour were monitored daily. Body weight, food, and fluid intake were monitored every week.

2-  Hematology and serum biochemical analyses. Blood samples were collected from the brachial or femoral veins:

CBC and serum chemistry were assessed on days 0, 7, 14, 28, 35, and 42 following blood sampling. Serum NAB titers were measured at days 0, 7, 14, 28, 35, and 42 following blood sampling. The CD4/CD8 ratio was assessed at days 0, 7, 14, 28, and 42 following blood sampling. Th1/Th2 antibody levels were determined at days 0, 7, 14, 28, and 42 following blood sampling.

3-  Other investigations:

X-ray examination was carried out on days 0, 14, and 42. Oropharyngeal swab specimens from vaccinated and control macaques were collected at days 0, 7, 14, and 42 and tested for viral RNA by RT-PCR.

The schedule of examinations is summarized in Table 2 as follows:

4-  Assessment of immunogenicity using viral microneutralization (VMN) test:

The viral microneutralization (VMN) test described in Alessandro et al. was used to assess the neutralizing activity of antibodies against SARS-CoV-2 in VERO cells, which are permissive for SARS-CoV-2 infection. Serum samples were heat‐inactivated for 30 min at 56°C and serially diluted twofold starting from 1:10. An equal volume containing 100 TCID50 of SARS‐CoV‐2 was added to each dilution, and the serum‐virus mixture was incubated for 1 h at 37°C in a humidified atmosphere with 5% CO2. After incubation, 100 µl serum-virus mixture was added in duplicate to a cell plate containing a semi‐confluent VERO monolayer, and the plates were then incubated for 4 days at 37°C in a humidified incubator with 5% CO2. The antibody titer of each serum sample was defined as the highest serum dilution that completely protected the cells from cytopathic effect (CPE) in at least half or at all wells, respectively [16].

5-  Hematologic and blood chemistry analysis:

 CBC was performed on blood samples collected in sterile ethylene diamine tetra acetic acid-containing vacutainer tubes using an automated cell counter (IDEXX Laboratories, USA). Additionally, blood samples were collected in plain tubes and sera were separated for automated analysis of serum glutamicoxaloacetic transaminase, serum glutamic-pyruvic transaminase, alkaline phosphatase (ALP), urea, and creatinine with the IDEXX analyzer system, and inflammatory markers (i.e., TNF-α, IL-6, and INF-ɣ) using specific ELISA kits (Origene, USA).

Statistical analysis

The Instat version 3 and GraphPad Prism version 5 (GraphPad Software Inc., USA) software packages were used for data analysis. Data were expressed as mean ± SD. Comparisons between different means were performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test to identify statistically significant differences between individual groups. Comparisons between different means of antibody titers at different time intervals were performed using two-way ANOVA. Statistical significance was defined when p < 0.05. Data were tested for normality.

Scheme 1: A general outline of the efficacy and safety study conducted from March 23, 2021, to May 04, 2021.

Results

In this study, four animals of each gender were subjected to immunization with escalating vaccine doses per animal. The kinetics of gender-specific total and differential counts of white blood cells, erythrocytes, and platelets were monitored at regular intervals post-immunization. In addition, T-cell responses and virus-neutralizing antibody titers were documented. Furthermore, procalcitonin, liver enzymes, and kidney function were recorded. Both data analysis and plots were generated using GraphPad PRISM version 5 software. The results were expressed as means ± standard deviations.

By presenting data for the highest dose of the vaccine (140 µg), the authors aim to highlight the significant results obtained. Although the other doses showed minimal post-immunization parameters, this particular dose gave the most appreciable results. The data presented is a testament to the efficacy of the vaccine and reinforces the importance of considering higher doses to achieve optimal results.

In general, monkeys showed no morbidity or mortality among all groups throughout the experiment. No abnormal clinical signs were observed in any macaque following immunization with the three doses.

Hematological and biochemical laboratory values were relatively normal at all the study endpoints compared to the control group. In several occasions, females showed higher both total and differential white blood cells counts than males in response to vaccination. This was hard to explain in the light of the used small sample size and needs to be confirmed on a bigger sample size (Fig. 1). Reticulocytes counts were higher among males than females at 7-42 days post-immunization. The peak reticulocytes percentage was seen for both genders at 7 days post-immunization (Fig. 2).

All platelets related parameters showed gradual increase post immunization and were relatively higher among females compared to males. The female-bias was hard to explain in the light of the used small sample size and needs to be confirmed on a bigger sample size (Fig. 3).

Where Th1/Th2 ratios were higher among males at early time points the opposite was seen at 42 days post vaccination. At 7 days post vaccination males showed slightly higher CD4/CD8 ratio than females but the opposite was seen at later time points. All test groups developed detectable neutralizing antibodies at all study intervals. The virus neutralizing antibodies titers were generally higher among females than males (Fig. 4).

There was a general female-bias in the levels of procalcitonin, alanine transaminase, alkaline phosphatase and bilirubin/ creatinine ratio at all time points post vaccination and the opposite was recorded for the creatinine levels. This was hard to explain in the light of the used small sample size and needs to be confirmed on a bigger sample size (Fig. 5).

RT-PCR testing confirmed the absence of virus genome amplification, indicating vaccine safety. X-ray evaluation showed no abnormalities for either test group.

Figure 1: Kinetics of the gender-specific total and differential white blood cells counts in rhesus macaque monkeys in response to immunization with EGYVAX. A total of 4 animals from each gender were immunized by 140 µg of the vaccine per animal. Both the total and differential counts were kinetically monitored at regular time intervals post immunization. Both data analysis and plots were done using the GraphPad PRISM version 5 software. Results were expressed as means ± standard deviations. In several occasions, females showed higher both total and differential counts than males in response to vaccination. This was hard to explain in the light of the used small sample size and needs to be confirmed on a bigger sample size.

Figure 2: Kinetics of the gender-specific erythrocyte counts and related parameters in rhesus macaque monkeys in response to immunization with EGYVAX. A total of 4 animals from each gender were immunized by 140 µg of the vaccine per animal. Both the total and differential counts were kinetically monitored at regular time intervals post immunization. Both data analysis and plots were done using the GraphPad PRISM version 5 software. Results were expressed as means ± standard deviations. Reticulocytes counts were higher among males than females at 7-42 days post immunization. The peak reticulocytes % was seen for both genders at 7 days post immunization.

Figure 3: Kinetics of the gender-specific platelets counts and related parameters in rhesus macaque monkeys in response to immunization with EGYVAX. A total of 4 animals from each gender were immunized by 140 µg of the vaccine per animal. Both the total and differential counts were kinetically monitored at regular time intervals post immunization. Both data analysis and plots were done using the GraphPad PRISM version 5 software. Results were expressed as means ± standard deviations. All platelets related parameters showed gradual increase post immunization and were relatively higher among females compared to males. The female-bias was hard to explain in the light of the used small sample size and needs to be confirmed on a bigger sample size.

Figure 4: Kinetics of the gender-specific T-cell responses and virus neutralizing antibodies titers in rhesus macaque monkeys in response to immunization with EGYVAX. Both data analysis and plots were done using the GraphPad PRISM version 5 software. Results were expressed as means ± standard deviations. Where Th1/Th2 ratios were higher among males at early time points the opposite was seen at 42 days post vaccination. At 7 days post vaccination males showed slightly higher CD4/CD8 ratio than females but the opposite was seen at later time points. The virus neutralizing antibodies titers were generally higher among females than males.

Figure 5: Kinetics of the gender-specific procalcitonin, liver enzymes and kidney function in rhesus macaque monkeys in response to immunization with EGYVAX. Both data analysis and plots were done using the GraphPad PRISM version 5 software. Results were expressed as means ± standard deviations. There was a general female-bias in the levels of procalcitonin, alanine transaminase, alkaline phosphatase and bilirubin/creatinine ratio at all time points post vaccination and the opposite was recorded for the creatinine levels. This was hard to explain in the light of the used small sample size and needs to be confirmed on a bigger sample size.