Hybrid immunity at 6 months after SARS-CoV-2 exposure in individuals from the community treatment program

doi:10.21203/rs.3.rs-1857122/v1

The death rate from SARS-CoV-2 infection (COVID-19) was lowered from 2021 to 2022, while the infection rate was increasing. Hybrid immunity, caused by vaccination and infection, including asymptomatic infection, may confer effective protection against death. We explored the combined effect of asymptomatic COVID-19 and hybrid immunity by studying T cell and antibody responses against SARS-CoV-2 in individuals treated in home healthcare service at 6 months after COVID-19.

Results

Asymptomatic COVID-19 was demonstrated in 24.4% of closed contacts. The levels of immunity were comparable between COVID-19 patients and close contacts. Anti RBD IgG against SARS-CoV-2 virus increased dose dependently to the numbers of vaccination, in contrast to T cell response that decreased at early period after vaccine booster.

Conclusions

Asymptomatic COVID-19 may not contribute to immunity against SAR-CoV-2 developed in vaccinated close contacts. However, when designing vaccine strategies, T cell exhaustion after multiple vaccinations is a point of concern.

Since early 2020, SARS-CoV-2 has spread globally and has become the most devastating pandemic in history. By mid-August 2021, Thailand faced delta variant emergence, with COVID-19 cases reaching more than 20,000 per day and more than 300 deaths daily (1). Multiple measures were implemented to curve the infection rate and reduce mortality, which included a nationwide vaccination program prioritizing populations at high risk of developing severe COVID-19 illness. The decline in new cases has been observed together with the higher adoption of vaccines in the public, especially with mRNA vaccines.

The Centers of Disease Control (CDC) reported that 66% of Americans were fully vaccinated and had a much lower mortality rate than the unvaccinated group (0.1 VS 0.76 per 100,000 population); however, mRNA was used as the primary vaccine option in the United States. In contrast, Chile had 91% of the population fully vaccinated but mostly with inactivated vaccine. The Chilean mortality rate was less different between the fully vaccinated and unvaccinated groups (0.41 VS 0.8 per 100,000 population). (2)

To date, more than 75% of the population in Thailand has been fully vaccinated, mostly with inactivated vaccine, and one-third received mRNA vaccine as a booster dose. The SARS-CoV-2 Omicron variant surged in 2022, and to date, 4 million people in Thailand have been infected (5% of the population). Again, new COVID-19 cases reached 50,000 per day; however, the death rate seems to be lower (120 per day) than the peak of the delta variant (300 per day). (1)

Many scientists expected herd immunity to be developed when most of the population gained immunization, either through vaccinations or past exposure to the virus. Nonetheless, the herd-immunity threshold was unlikely to be met because of vaccine hesitancy and the emergence of new variants. Long-term prospects for the pandemic may be the influence of COVID-19 becoming an endemic disease and contemplating a new normal that does not include herd immunity. Interestingly, the evidence from the United Kingdom study (3) revealed "hybrid immunity", the combined efficacy of vaccination and SARS-CoV-2 infection, is long-lasting and confers highly effective protection against symptomatic disease for at least six to eight months after vaccination before the advent of Omicron.

We observed a lower infection rate in close contacts of COVID-19 patients who received favipiravir-based treatment in a home-based setting during the delta outbreak in Bangkok. During the outbreak, more than 30,000 cases were treated at home, with limited social distancing due to space unavailability and the number of infected cases in the same household. We hypothesized that the lower death rate of COVID-19 patients may be due to herd immunity in terms of hybrid immunity, in addition to the lower virulence of Omicron. Moreover, evidence revealed that (memory) T cell immunity against COVID-19 developed in close contact persons (asymptomatic cases) (4). These occult infections may contribute to some degree of herd immunity in society. Therefore, we studied T cell immunity and antibody responses against SARS-CoV-2 in patients and close contacts who were registered in the Bangkok home healthcare service at 6 months after SARS-CoV-2 exposure.

Subjects

This observational study enrolled 79 participants from 15 families who were registered in a database of the Bangkok home healthcare service (one family was randomly selected per district area) between 1 and 31 August 2021. Thirty-four cases were individuals who recovered from COVID-19 for at least 4 weeks before enrollment, while 45 cases were close contacts enrolled according to inclusion criteria (at least 1 asymptomatic case per family, with negative antigen test after COVID patients in family were diagnosed, living in the same accommodation following regulation of national health policy). The calculated sample size was 90 cases, based on the prevalence of asymptomatic COVID-19 in the Chinese population (4), with 80% power to detect asymptomatic infection. Peripheral blood (15 ml) was collected after written informed consent was obtained from each participant. The study was approved by the ethics committee, Ramathibodi Hospital, Mahidol University (MURA2021/923) and Bangkok Hospital (BHQ-IRB 2021-11-34) according to the Declaration of Helsinki, the Belmont Report, CIOM Guidelines and the International Conference on Harmonization in Good Clinical Practice (ICH-GCP).

Materials and Reagents

A FACSLyric™ (Becton Dickinson, USA) flow cytometer was employed for cytokine detection, and Beckman Coulter’s FC 500 series (Beckman Coulter, USA) was employed for CBC determination. The Alinity and ARCHITECT Systems were used for IgG to SARS-CoV-2 level and EUROIMMUN Analyzer I for ELISA reader. The incubator, centrifuge, vortex mixer and automatic cell counter were from Thermo Fisher Scientific Inc. (USA). Phosphate-buffered saline pH 7.4 (PBS) was from Sigma-Aldrich (USA), heparin and EDTA tubes from Becton Dickinson (USA), and RPMI-1640 medium from Life Technologies (USA).

The enzyme-linked immunosorbent spot (ELISpot) assay T-SPOT.

Fresh heparinized whole blood samples (10 mL) from volunteers were isolated for peripheral blood mononuclear cells (PBMCs) using Sepmate PBMC isolation tubes (STEMCELL Technologies Inc., Canada). The heparinized blood was diluted with RPMI medium (1:1) in Sepmate PBMC isolation tubes and centrifuged at 1700 g at 20°C for 20 minutes. The separate PBMCs were washed twice with PBS and recentrifuged at 500 g for 5 minutes at 4°C. PBMCs (2.5x105 cells) were added to 96-well plates precoated with an anti-IFN-g antibody of the T-SPOT®. COVID test (Oxford Immunotec, Ltd., UK). The plate stimulated each sample composed of four wells with two antigen antigens against Spike (S) protein and Nucleocapsid (N) protein, membrane glycoprotein (M) and ORF1ab region of RNA-dependent RNA (O) of SARS-CoV-2 alpha variant; phytohemagglutinin (PHA) and medium alone were used as the positive and negative controls, respectively. Plates were maintained overnight at 37°C in a 5% CO2 humidified atmosphere, washed with phosphate-buffered saline and developed using an anti-IFN-g antibody conjugate and substrate to detect the presence of secreted IFN-g. Spot-forming cells (SFCs) were counted with an automated ELISpot reader (CTL Analyzers, Cleveland, OH, USA). SFC less than 10 per 250,000 cells was represented as normal background according to manual recommendation that was comparable to the lowest quartile of SFC in COVID patients in this cohort.

IgG level of SARS-CoV-2 in the receptor binding domain (RBD)

Human EDTA plasma samples were measured to quantitatively determine IgG antibodies against the spike receptor-binding domain (RBD) to SARS-CoV-2, which used the SARS-CoV-2 IgG II Quant assay on the Alinity and ARCHITECT I Systems. The chemiluminescent reaction is calculated as a relative light unit (RLU) and expressed as a calculated index (S/C). The SARS-CoV-2 IgG II Quant assay cutoff is 50 AU/mL, and those greater than 50 AU/mL are interpreted as positive.

ELISA to detect SARS-CoV-2-specific neutralizing antibodies

Human EDTA plasma samples were diluted 1:5 in sample buffer, which was performed according to the manufacturer’s instructions for The Euroimmun SARS-CoV-2 NeutraLISA (Euroimmun AG, Lübeck, Germany). In brief, 100 μl of the diluted sample, control, or blank was added per well and incubated at 37 °C for 1 h. The automatic machine washed the plate 3 times with wash buffer; then, 100 μl of enzyme conjugate was added and incubated at RT for 30 min. After the washing cycle, 100 μl of substrate solution was added, and the plate was incubated at RT for 15 min. Finally, 100 μl of stop solution was added per well, and the absorption at 450 nm was measured using a EUROIMMUN Analyzer I. Samples were analyzed in a single replicate. The percent inhibition (%IH) was calculated as follows: 100% - ((extinction of sample x 100%)/extinction of blank). The Euroimmun recommends interpreting results as follows: %IH <20: negative; %IH >20 to <35: borderline; %IH >35: positive.

Statistical analysis

Descriptive results are presented as the median (interquartile range) and percentage ± SD. Corresponding inferential comparisons were calculated using the t-test or Mann-Whitney U test using GraphPad Prism 9.4.0.673.

Asymptomatic COVID-19 infection in close contacts

During the SARS-CoV-2 exposure period, COVID-19 patients and close contacts in the study cohort were living in the same accommodations. There were 15 families in the cohort that comprised 11 individuals (median, range 3-30) per family in a living space of 200 m²(median, range 30-400 m²). Most of the participants were female (58%) and younger than 60 years (91%), and their body mass index was less than 30 (81%). Comorbidities defined as risks of severe COVID infection were described in one-fifth of the population (26.5% in COVID patients and 15.5% in close contacts). (Table 1) Our data showed that the T cell response against the S antigen in close contacts was comparable to that in recovered COVID-19 patients. (Figure 1) Since 75% of participants were fully vaccinated, the T cell response against the S antigen in this population may be the result of previous infection or vaccination. Therefore, we analyzed the T cell response against NMO antigens and found a T cell response (at a cutoff of 10 SFCs/250,000cells) in 11 of 45 cases (24.4%) of close contacts that was assumed to be evidence of previous infection. (Figure 1) However, a history of vaccination with a whole virus molecule (inactivated vaccine) may be a confounding factor. One-fourth of the participants were fully vaccinated with inactivated vaccine at 3.5 months before enrollment. To explore this issue, we analyzed the history of vaccination in 11 close contacts and found only 2 cases that received inactivated vaccine. Then, the T cell response against the NMO antigen in these cases was most likely due to occult or asymptomatic COVID infections.

Hybrid immunity against SARS-CoV-2 in recovered COVID-19 patients and close contacts.

In this studied population, the percentage of neutralizing antibody (%NT) was correlated with the levels of RBD IgG (Figure 2). At 6 months post SARS-COV-2 exposure, COVID patients without vaccination had a very low level of RBD IgG, comparable to close contacts. However, the T cell and antibody response against SARS-COV-2 in close contacts with asymptomatic infection was not different from other cases of close contacts (Figure 3).

After vaccination, increments of RBD IgG levels were seen in a dose-dependent manner to numbers of vaccinations in both groups. The T cell response did not increase in the same manner as RBD IgG. We observed a decreased T cell response against S antigen (S ELISPOT) after the booster vaccine (3^rd & 4^th dose). (Figure 4) The T cell response in individuals who received 3 or 4 doses of COVID vaccine was comparable to that in individuals without any vaccination. To explore vaccine type as a confounding factor, we depicted types of vaccines in the study population and found that 75% of participants were fully vaccinated with inactivated vaccine or viral vector vaccine (3.5 months before enrollment), while less than one-third of them received the 3^rd or 4^th booster dose with mRNA vaccine (1 month before enrollment). (Figure 5) The number and type of vaccines in patients and close contacts are described in the demographic data (Table 1).

At 6 months after SARS-CoV-2 exposure, we observed a T cell response against NMO antigens (interferon release assay) (5) in 11 of 45 close contacts. The rate of occult or asymptomatic COVID infection in the study population was 24.4%, which was concordant with a previous study that reported 16%-26% asymptomatic COVID infection in close contacts explored by (ex vivo) CD4 and CD8 memory T cell responses (4). Nonetheless, we observed comparable immunity development after vaccination between COVID-19 patients and close contacts, as shown by RBD IgG antibody levels and T cell response against S antigen after the second dose of vaccination. This result revealed that COVID immunity in the participants was boosted mostly by vaccination but not COVID infection in terms of hybrid immunity. In addition, we observed comparable antibody and T cell responses to SARS-CoV-2 in close contacts with asymptomatic infection (defined by T cell response against NMO antigens) and other closed contact cases. Nonetheless, we did not have long-term observational data on the reinfection rate after SARS-CoV-2 exposure, and none of the participants were reinfected with SARS-CoV-2 at enrollment (3).

In this study population, most of the participants received a heterogeneous prime-boost COVID vaccine comprising inactivated vaccine, viral vector vaccine and mRNA vaccine. A previous report revealed good efficacy of a heterogenous prime-boost vaccine in terms of neutralizing antibodies and T cell responses (6). However, in this study, the neutralizing capacity of RBD IgG was tested against wild-type SARS-CoV-2. Recently, a report from Hong Kong showed a reduced neutralizing capacity of RBD IgG against the omicron variant compared to wild-type SARS-CoV-2 in different strategies of vaccination with or without previous COVID infection (7). Moreover, after a booster dose of the COVID vaccine, we observed a lower T cell response against the spike protein, in contrast to higher RBD IgG levels, which is discordant with a previous study that revealed correlations between T cell responses to spike and nucleoprotein/membrane proteins and peak antibody levels (5). In addition, a lower T cell response (S ELISPOT) was observed at 1 month after the booster vaccine, which is discordant with a previous study that revealed that CD4 + responses were typically detectable by day 8 after priming, peaked soon after vaccine boost, and then fell to preboost levels after 4 months (8). Nonetheless, after a short period of T cell exhaustion, as observed in our study, the T cell response may recover at 3 months after a booster dose of vaccine, as shown in a preliminary report from F Zuo et al that revealed an antibody response and T cell response against the SARS-CoV-2 omicron variant after heterogeneous immunization with inactivated vaccine followed by mRNA booster (9).

Individuals may exhibit wide variation in cellular and humoral immune responses during primary viral infection, with some patients displaying imbalanced viral-specific B cell and T cell immunity, especially individuals who suffer severe and long-lasting symptoms (10). Since most of our study population was boosted with an mRNA vaccine, it was also possible that the mRNA vaccine hampers T cell function after vaccination (11, 12). Nonetheless, this result raised our concern about T cell exhaustion after multiple doses of vaccine booster, especially in an early period after a short interval of vaccine booster. This observation might explain the evidence of herpes zoster reactivation after receiving the COVID vaccine due to transient lymphopenia (13). Therefore, multiple booster doses of COVID vaccine should be used with considerations in the case of immunocompromised hosts that have poor T cell response and closed monitoring for infection, especially early after a booster dose of vaccine. Moreover, in the current situation with decreased neutralizing antibodies against the omicron variant (9), a T cell-based vaccine may be needed (10, 14).

Even with limitations in the study, such as the small number of participants, long duration after SARS-CoV-2 exposure (6 months) and immune response confounded by vaccination, this study revealed the antibody and cellular response in both symptomatic and asymptomatic COVID infection that helped to define COVID immunity in society and design a vaccine strategy and may predict long-term protection in the context of hybrid immunity after booster vaccination.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Acknowledgement

This study was financially supported by the Bangkok Health Research Center, Bangkok Dusit Medical Service Plc.

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Table 1 Demographic data

Demographic data	Total number (%)	COVID patients (%)	Close contacts (%)
SAR CoV viral exposure	79	34	45
Gender
Female	46 (58.2)	23 (67.6)	23 (51.1)
Age
18-60 yrs	72 (91.2)	30 (88.2)	42 (93.3)
> 60 yrs	7 (8.8)	4 (11.8)	3 (6.7)
BMI
<25	44 (55.7)	14 (41.2)	30 (66.6)
25-30	20 (25.3)	13 (38.2)	7 (15.5)
>30	15 (19)	7 (20.6)	8 (17.9)
Risk factors of severe COVID infection	15 (18.9)	9 (26.5)	7 (15.5)
Coronary arterial disease (CAD)	3	1	2
Diabetes mellitus (DM)	7	6	1
Chronic lung disease	1	0	1
Chronic kidney disease	1	1	0
Cancer	1	1	0
DM with CAD	1	0	1
DM with asthma	1	0	1
Numbers of vaccine
0	13 (16.4)	4 (11.8)	9 (20)
1	7 (15.5)	4 (11.8)	3 (6.7)
2	35 (44.3)	13 (38.2)	22 (48.9)
3	18 (22.8)	9 (26.4)	9 (20)
4	6 (1)	4 (11.8)	2 (4.4)
Type of vaccine
1st dose	66	30	36
Inactivated vaccine	23 (34.8)	9 (30)	14 (38.9)
viral vector vaccine	39 (59.1)	18 (60)	21 (58.3)
mRNA vaccine	4 (6.1)	3 (10)	1 (2.8)
2nd dose	59	34	25
Inactivated vaccine	19 (32.2)	16 (47)	3 (12)
viral vector vaccine	32 (54.2)	12 (35.3)	20 (80)
mRNA vaccine	8 (13.6)	6 (17.7)	2 (8)
3rd dose	24	9	15
viral vector vaccine	7 (29.2)	4 (44.5)	3 (20)
mRNA vaccine	17 (70.8)	5 (55.5)	12 (80)
4th dose	6	4	2
viral vector vaccine	1 (16.6)	1 (25)	0 (0)
mRNA vaccine	5 (83.4)	3 (75)	2 (100)
Diagnostic test for COVID infection
Antigen rapid test		33
RT-PCR		13
Treatment
Favipiravir		28
Remdesivir		2
Cortocosteroids		4
None		5
Refer to hospital		8
Accommodation
Living areas
30-60 m2	13 (16.5)
80-200 m2	30 (38)
200-400 m2	36 (45.5)
Numbers of family member
3-5	32 (40.5)
6-15	17 (21.5)
16-30	30 (38)

Competing interest reported. Pongtorn Kietdumrongwong is employed by the funder (BDMS) of this research, as the director of research. He hold share in BDMS for personal purchasing. He also employed by the government office for emergency medicine as an expert in standard and quality improvement.

Parawee Chevaisrakul and other co-authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Hybrid immunity at 6 months after SARS-CoV-2 exposure in individuals from the community treatment program

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Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Tables

Additional Declarations

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