A survey on some immune indicators in the group of vietnamese children who did not respond to rotavac vaccine

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A SURVEY ON SOME IMMUNE INDICATORS 
IN THE GROUP OF VIETNAMESE CHILDREN 
WHO DID NOT RESPOND TO ROTAVAC VACCINE 
Nguyen Minh Hai1, Phung The Hai³, Do Khac Dai³ 
Pham Minh Dam1, Pham Ngoc Hung², Nguyen Dang Dung³ 
SUMMARY 
Objectives: To investigate the expression of beta 7 integrin on B and T lymphocytes using a 
flow cytometry system. Subjects and methods: We analyzed immunological parameters such 
as B and T lymphocyte population as well as B and T cell subpopulations expressing beta 7 
integrin molecules by flow cytometry on the two groups of children with and without response to 
the Rotavirus vaccine. Results: The number of peripheral blood lymphocytes of the non-response 
group was statistically significantly lower than that of the response group ( ± SD: 4.89 ± 1.71 vs 
5.77 ± 1.46 million/mL, p = 0.04). The number of T cells (CD3+) of the non-response group was 
statistically significantly lower than that of the response group ( ± SD: 1.65 ± 0.61 vs 2.05 ± 
0.64 million/mL, p = 0.018). The frequency and number of T-cells expressing beta 7 integrin of 
the non-response group were statistically significantly lower than those of the response group 
(Frequency ( ± SD): 17.48 ± 2.89 vs 19.44 ± 3.74%, p = 0.029; Quantity ( ± SD): 0.87 ± 0.41 vs 
1.13 ± 0.37 million/mL, p = 0.014). There was no statistically significant difference between 
2 groups for B-cell-related parameters in peripheral blood such as the number and frequency of 
total B cells and the B-cell subpopulation expressing beta 7 integrin in peripheral blood. 
Conclusion: The group of children not responding to the Rotavirus vaccine was statistically 
significantly lower than that of the responding group for the following parameters: Total lymphocyte 
count; frequency and number of the subpopulation of T-lymphocytes expressing 7 beta integrin 
in peripheral blood. 
* Keywords: Rotavirus vaccine; Beta 7 integrin; β7- integrin; B lymphocytes; T lymphocytes. 
INTRODUCTION 
Rotavirus is a common cause of 
diarrhea among children globally as well 
as in Vietnam [1]. Currrently, orally live-
attenuated Rotavirus vaccines have been 
proven effective and widely used [2]. 
However, there is a proportion of non-
responders to these vaccines, particularly 
in low-income countries such as in Africa 
and Asia, where the efficacy ranges from 
51% to 64% on average [3, 4, 5]. Factors 
influencing vaccine efficacy may include: 
immune status of the body, general 
nutritional status, nature of circulating 
rotavirus strains, intestinal microbiota, etc. 
1Department of Exams Accreditation and Medical Education Quality Assurance, Vietnam Military 
Medical University 
2Department of Epidemiology, Vietnam Military Medical University 
³Department of Immunology, Vietnam Military Medical University 
Corresponding author: Nguyen Minh Hai (minhhaiym@gmail.com) 
 Date received: 03/02/2021 
 Date accepted: 10/4/2021 
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Based on a clinical trial study of ROTAVAC 
vaccine in children aged 6 - 8 weeks, we 
conducted this study: To investigate a 
number of factors affecting the 
immunogenicity of the rotavirus vaccine in 
Vietnamese children. 
After oral vaccination of Rotavirus 
vaccine, viral antigens were captured and 
presented by antigen-presenting cells 
(APCs). The APCs then migrate to the 
intestinal secondary lymphoid organs or 
mesenteric lymph nodes to present viral 
antigens to specific B and T cells, 
resulting in activation of the humoral 
immune response (antibody production) 
and the cell-mediated immune response 
(helper T and cytotoxic T cells) specific to 
Rotavirus. The specific B and T cells then 
differentiate, proliferate, and move into 
the blood, before moving through the 
intestinal capillaries and retaining in the 
intestine. The gut-homing process of 
specific B and T lymphocytes depends on 
the expression of an important molecule, 
beta 7 integrin. Beta 7 integrin can 
combine with another molecule, alpha 4 
integrin on lymphocytes to specifically 
bind to MAdCAM-1 molecule (expressed 
only on intestinal capillary endothelial cells). 
After penetrating the intestinal capillaries, 
beta 7 integrin can also combine with 
alpha E integrin molecule to specifically 
bind E-cadherin molecule (expressed only 
on intestinal epithelial cells), by which 
lymphocytes B or T can be anchored in 
the intestine to perform their task [6]. 
We hypothesized that there might be a 
difference in the expression of beta 7 
integrin molecule on the surface of B and 
T lymphocytes (which plays an important 
role in the humoral immune response) 
between the two groups of children with 
response and non-response to Rotavirus 
vaccine. Therefore, this study was carried 
out to investigate the expression of beta 7 
integrin on B and T lymphocytes using a 
flow cytometry system. 
SUBJECTS AND METHODS 
1. Subject, time, place of the study 
- Study subjects: 58 children receiving 
ROTAVAC® vaccine and having been 
tested for IgA with the results of antibody 
titration after vaccination who were 
divided into two groups: Group 1 (n = 30): 
With very low initial antibody titres and/or 
the ratio after/before vaccination below 
1.5. Group 2 (n = 28): With the relatively 
high initial antibody titres and/or the ratio 
after/before vaccination ≥ 3 [7]. 
- Location: The study was conducted in 
Hung Ha district, Thai Binh province. 
- Time: From June 2020 to July 2020. 
After using ROTAVAC® vaccine 
(3 doses enough), children were given 
2 - 3 mL of EDTA blood to analyze some 
parameters of immune cells by flow 
cytometry or total blood count. Collected 
samples were coded and double-blindly 
analyzed. 
2. Methods 
- A protocol of evaluating lymphocytes 
with a flow cytometry system: 
50 µL of peripheral blood of each study 
subject was incubated with fluorescent 
antibodies (Abs) including anti-CD45 
antibody with PerCP (anti-CD45-PerCP), 
anti-CD3 antibody with FITC (anti-CD3-
FITC), anti-CD19 antibody with APC 
(anti-CD19-APC) and anti-beta 7 integrin 
with PE (anti-beta 7 integrin-PE). In addition, 
we used isotype control antibody 
conjugated with PE (a clone similar to the 
clone of anti-beta 7 integrin) to distinguish 
negative or positive populations with Beta 
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we used isotype control antibody 
conjugated with PE (a clone similar to the 
clone of anti-Beta 7 integrin) to distinguish 
negative or positive populations with Beta 
7 integrin (Isotype control-PE). All antibodies 
were manufactured by Biolegends. The 
incubation condition was 30 minutes at 
room temperature (25°C). 
Then, use 450 µL of red blood cell lysis 
buffer 1X (Biolegends) for each of the 50 
µL of the stained peripheral blood 
samples, and incubate it for 15 minutes at 
room temperature. 
Next, the samples were analyzed by 
the ACEA Novocyte flow cytometry system. 
- Total blood count: 
Total white blood cell counts were 
assessed with a total blood count machine. 
3. Statistical analysis 
We tested the statistical differences of 
the two groups by the student's T-test. 
RESULTS AND DISCUSSION 
With the 3-dose course of Rotavirus vaccine, some children did respond and some 
children did not respond to Rotavirus vaccine. Peripheral blood from 2 groups of response 
(the specific antibody concentration increased 3 times compared to the antibody 
concentration before the course) and non-response (specific antibody concentration 
was 1.5 times less than pre-course antibody concentration) were collected. 
Figure 1: The procedure for analyzing peripheral lymphocyte parameters (A) and 
how to analyze the data on a flow cytometry system (B). 
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To count the percentage and number of B or T lymphocytes expressing beta 7 integrin, 
we stained the peripheral blood cells with CD45-specific antibody (expressed on all 
immune cells), CD3-specific antibody (expressed on T cells), CD19-specific antibody 
(expressed on B cells), and beta 7 integrin-specific antibody. Figure 1B depicts how we 
analyzed the stained blood samples by using a flow cytometer. In addition, the blood 
samples were also analyzed by the total blood count to obtain lymphocyte count. 
Figure 2: The numbers and frequency of T and B lymphocytes in peripheral blood of 
two groups responding and not responding to Rotavirus vaccine. 
We found that the number of peripheral blood lymphocytes of the non-response 
group was statistically significantly lower than that of the response group ( ± SD: 4.89 ± 
1.71 vs 5.77 ± 1.46; million/mL); p = 0.04 (figure 2A). In addition, the number of T cells 
(CD3+) in the non-response group was also statistically significantly lower than that in 
the response group ( ± SD: 1.65 ± 0.61 vs 2.05 ± 0.64 million/mL; p = 0.018) (figure 2C). 
However, the frequency and number of B cells between the two groups were not 
statistically different (figure 2D, 2E). 
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Figure 3: The number and frequency of T and B lymphocytes expressing beta 7 integrin in 
peripheral blood of two groups of response and non-response to Rotavirus vaccine. 
We continued to evaluate the expression 
of beta 7 integrin on B and T lymphocytes. 
The data showed that the frequency and 
number of integrin beta 7-expressing 
T-cells in the non-response group were 
statistically lower than those in the 
response group (Frequency: ( ± SD): 
17.48 ± 2.89 vs 19.44 ± 3.74%; p = 0.029; 
Quantity ( ± SD): 0.87 ± 0,41 vs 1.13 ± 
0.37 million/mL, p = 0.014) (figure 3A, 3B). 
However, we observed no difference in 
the frequency and number of beta 7 
integrin B-cells between the two groups 
(figure 3C, 3D). 
Contrary to our initial expectation, the 
Rotavirus-specific IgA antibody concentration 
between the responders and the non-
responders was not similar, there might 
be some difference in B-lymphocyte 
populations between two groups. Our data 
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showed that B cells between the two 
groups showed no difference in the 
frequency and number of total B cells as 
well as the beta 7 integrin expressing 
B-cell subpopulation (figures 2D, 2E, 
3C, 3D). Interestingly, we observed a 
difference in the beta 7 integrin 
expressing T subpopulation, in which the 
non-response group showed lower 
frequency and number than the response 
group. (figures 3A, 3B). Although the role 
of secreting antibodies specific to Rotavirus 
(IgA, IgG) is predominant due to the 
specific B cells, the specific T cells may 
also play a role in assisting B cell 
proliferation and differentiation. This 
phenomenon occurs in the secondary 
lymphoid organs in the intestine such as 
the Payer patches or the mesenteric 
lymph nodes. The difficulties in accessing to 
these human tissues hinder understanding 
of the specific molecular biology 
mechanisms behind the vaccine 
unresponsiveness. Our results suggest 
further studies on the correlation between 
expression of the beta 7 integrin molecule 
on T cells and the specific antibody 
immune response. On the other hand, 
many studies have shown the important 
role of the vitamin A/rentinoic acid axis 
(a product of vitamin A metabolism via an 
enzyme retinal dehydrogenase in antigen-
presenting cells) in the expression of 
molecules that are important for T 
lymphocytes to return to the intestine 
such as beta 7 integrin or CCR9 on T 
cells, as well as MAd-CAM-1 on intestinal 
vascular endothelial cells, and CCL25 
(bound to CCR9) by the intestinal mucosa 
secreted [8, 9]. Our study suggests that 
vitamin A deficiency in young children 
should be further explored when using 
oral vaccines absorbed through the 
intestinal mucosa. 
CONCLUSION 
The group of children who did not 
respond to the Rotavirus vaccine showed 
statistically significantly lower parameters 
compared to the response group 
including total lymphocyte count; 
frequency and number of subpopulation 
of T-lymphocytes expressing an 7-beta 
integrin in peripheral blood. 
There was no statistically significant 
difference between the two groups for 
peripheral blood B-cell-related parameters 
such as the number and frequency of 
total B cells and the B-cell subpopulation 
expressing beta 7 integrin. 
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