An Early and Preliminary Assessment of the Clinical Severity of the Emerging SARS-CoV-2 Omicron Variants in Maharashtra, India

Background: The SARS-CoV-2 Omicron variants BA.2.74, BA.2.75, and BA.2.76 have appeared recently in India and have already spread to over 40 countries. They have acquired additional mutations in their spike protein compared to BA.2, branching away on the SARS-CoV-2 phylogenetic tree. These added mutations have raised concerns about the impact on viral pathogenicity, transmissibility, and immune evasion properties of the new variants. Material and methods: A total of 990 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) positive SARS-CoV-2 samples, with a cycle threshold value (Ct) less than 25, were processed for SARS-CoV-2 whole genome sequencing between June 3, 2022 to August 7, 2022. All corresponding demographic and clinical data were recorded and analyzed using Microsoft® Excel. Results: Out of 990 samples sequenced, BA.2.75 (23.03%) was the predominant Omicron sublineage, followed by BA.2.38 (21.01%), BA.5 (9.70%), BA.2 (9.09%), BA.2.74 (8.89%) and BA.2.76 (5.56%). A total of 228 cases of BA.2.74, BA.2.75, and BA.2.76 were contacted by telephone, of which 215 (94.30%) were symptomatic with mild symptoms, and 13 (5.70%) had no symptoms. Fever (82.02%) was the most common symptom, followed by cough (49.12%), cold (35.97%), fatigue (27.19%), headache (21.05%), and myalgia (20.61%). Of the 228 cases, 195 (85.53%) cases recovered at home, and 33 (14.47%) required institutional quarantine. Recovery with conservative treatment was observed in 92.98% of cases, while 4.83% required additional oxygen therapy. Only three (1.32%) cases had poor outcomes resulting in death, and the remaining 225 (98.68%) survived. Among the 228 cases, 219 (96.05%) cases were vaccinated with the COVID-19 vaccine; of these, 72.60% had received both doses, 26.03% had also received the precautionary booster dose, while 1.37% were incompletely vaccinated with a single dose of vaccine. Conclusion: The current study indicates that the three BA.2 sublineages are causing mild disease in India. However, BA.2.75 has key mutations that are notable for accelerated growth and transmission and require close and effective monitoring.

The first Omicron variant, B.1.1.529 was first reported from South Africa on November 24, 2021 [2] and was designated as a Variant of Concern (VoC) on November 26, 2021 by World Health Organization. By late A set of individual-level data was obtained, corresponding to the samples received from various RT-PCR laboratories sending samples for sequencing. Each sample's unique identification number (ICMR ID) was also recorded. Additional information on the presence of any symptoms, hospitalization, treatment, comorbidities, and vaccination status was collected via a telephonic interview with each patient.

Statistical analysis
All demographic and clinical data were recorded and analyzed using Microsoft® Excel.

Results
Between June 3, 2022 and August 7, 2022, 990 RT-PCR-positive SARS-CoV-2 samples were processed and sequenced at BJGMC, Pune. The study population included cases from all age groups with a median age of 36 years. The male-to-female ratio was 1.39:1. Table 1 shows the geographical distribution of the sequenced samples.

Geographical Area Number (%)
Pune 792 (80%)      India, on the other hand, did not see an exponential increase in cases due to these two lineages. Based on the sequences deposited on GISAID, the BA.5 and BA.4 Omicron lineages continue to dominate globally, with a weekly prevalence of 69.6% and 11.8%, respectively [17]. However, it is interesting to note that, in India, the prevalence of BA.5 and BA.4 lineages is 9% and less than 0.5% among the sequences deposited on GISAID, respectively [13]. This probably could be as both Delta and BA.4/ BA.5 share the L452R mutation on the receptor-binding domain (RBD) of spike protein, the convalescent sera from Delta infection may contain L452R-specific neutralizing antibodies, which could have impaired the BA.4/ BA.5 transmission in India [18]. Also, BA.2.75 has shown 57-fold higher binding affinity to ACE2 receptors when compared with BA.5, accounting for its higher transmissibility [19].

FIGURE 2: Lineage prevalence in India over the last 90 days
This graph is generated and taken from Outbreak.info as of September 9, 2022 [13].
The SARS-CoV-2 virus has evolved rapidly, adapting to its human hosts by developing mutations over time and resulting in the emergence of new variants.  (Figure 3) [9]. The effect of the mutations in the RBD region on the virus-host interactions is described in Table 6 [20]. R493Q and N460K mutations increase the ACE-2 receptor affinity and surface RBD expression [21,22]. On the other hand, mutations G446S and G339H decrease the ACE-2 affinity and RBD expression. Such adaptive mutations can alter the pathogenic potential of the virus.

sublineages and their effect on virus-host interaction
The data represented here are generated using Jbloomlab.github.io [20] Table 7 compares the growth advantage of the common variants in India. These estimates reflect the advantage of the new variants compared to the cocirculating variants. The relative growth of a variant can be explained by three mechanisms: increased transmissibility, infectious duration, and immune evasion. The BA.2.75 sublineage has a relative growth advantage of 77% per week, with 0.08 as an assumed logistic growth rate per day. It has a 42% increase in transmissibility [23]. The spike protein mutations D339H, G446S, N460K, and R493Q allow BA.2.75 to escape neutralization by antibodies produced against different RBD epitopes in BA.2 [21]. Figure 4 shows the effect of antibodies elicited during the pre-Omicron and Omicron BA.1 period on the BA.2.75 variant. The mutations in the RBD region confer an escape fraction of 0.45 [24]. These features of BA.2.75 indicate that it might outcompete BA.4/ BA.5 and can become a potential risk to global health. Therefore, the spread and frequency of these sublineages in India and countries outside India require close monitoring through sustained genomic and clinical surveillance as they possess key mutations that are notable for their accelerated growth and extensive geographical distribution.   The current study indicates that these BA.2 sublineages caused mild disease with reduced need for hospital admission. In animal models, BA.2.75 replicated more efficiently in the lungs of hamsters than other Omicron variants causing focal pneumonia characterized by patchy inflammation in alveolar regions. These findings suggest that the Omicron subvariant BA.2.75 can cause severe respiratory disease and may affect the clinical outcome in infected humans [30]. However, it is still unclear to what extent the intrinsic virulence of the virus and the immunity due to vaccination or previous infections could have contributed to mild disease in India. Further, data from other clinical settings will be essential to assess the behavior of these BA.2 sublineages in countries with different levels of previous infections and vaccination.

Growth
The fact that subvariant BA.2.75 contains mutations greater than BA.2 and BA.4/BA.5 raises concern regarding the possibility that it might have significantly reduced sensitivity to therapeutic monoclonal antibodies and antibodies developed by vaccination/natural infection. It is important to note that these subvariants have emerged when the world is about to achieve global immunity against the SARS-CoV-2 virus through various vaccines available for COVID-19. India has conducted 200 crore vaccinations and has vaccinated 73.02% of its population with at least one dose of vaccine and 66.85% of the population with two doses of vaccine. Around 7.67% of the total population has received the precautionary dose [31]. Various studies on the evasion of neutralizing antibodies have found that the Omicron subvariant BA.2.75, which has a local growth advantage in India, is 1.8 and 1.1 times more resistant to sera from vaccinated individuals than BA.2 and BA.2.12.1, respectively. However, it is 0.6 times more sensitive than BA.4/BA.5. It has increased resistance to class 1 and 3 monoclonal antibodies but is sensitive to class 2 monoclonal antibodies like Casirivimab (REGN10933), Tixagevimab (COV2-2196) and S2E12. This increased resistance may be due to the spike protein's G446S and R460K mutations. It is also 3.7 times more resistant to Bebtelovimab, the only monoclonal antibody potent against all Omicron subvariants [22]. Another study in a small sample of plasma from post-vaccination Delta infection shows that the BA.2.75 subvariant is more immune evasive than the BA.4/BA.5 lineages in the Delta-stimulated immune background, which probably may explain why BA.2.75 has a growth advantage over BA.4/BA.5 in India [18]. Also, a study found that BA.2.75 has a higher resistance to BA. 5-induced immunity. This property may make BA.2.75 variant spread efficiently in areas where BA.5 has been widely circulating [32]. Close monitoring and following these variants effectively and investigating their development as early as possible is crucial.

Conclusions
To conclude, this study provides essential and early evidence of the severity of the disease in patients infected with BA.2.74, BA.2.75, and BA.2.76 sublineages. Most individuals suffered from mild illnesses like fever, cough, cold, fatigue, and headache. Currently, there is no evidence of an increased risk of hospital admission or severe disease due to these sublineages in India. Despite these second-generation variants being detected only recently, they have the potential to be successfully transmitted across several countries due to the presence of critical mutations and significant growth advantages. The ability of the SARS-CoV-2 virus to evolve continuously and achieve increased transmission and immune evasion reinforces the importance of vaccination and sustained epidemiological surveillance to detect emerging new variants.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethics Committee, BJGMC, Pune issued approval BJGMC/IEC/Pharmac/0721233-233. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.