key: cord-0783581-eivr6krq
authors: Pauvolid-Correa, A.; Costa Caetano, B.; Machado, A. B.; Araujo Ferreira, M.; Valente, N.; Keren, T.; Geraldo, K.; Motta, F.; Goncalves Veloso dos Santos, V.; Grinsztejn, B.; Mendonca Siqueira, M.; Resende, P. C.
title: Humoral immune response of patients infected by earlier lineages of SARS-CoV-2 neutralizes wild types of the most prevalent variants in Brazil
date: 2022-01-28
journal: nan
DOI: 10.1101/2022.01.24.22269379
sha: e1f0f63798b3d5453f013844e5cfc1e4dd22e8a8
doc_id: 783581
cord_uid: eivr6krq

In the present study, serum samples of 20 hospitalized COVID-19 patients from Brazil who were infected by the earlier SARS-CoV-2 lineages B.1.1.28 and B.1.1.33, and by the variant of concern (VOC) Gamma (P.1) were tested by plaque reduction neutralization test (PRNT90) with wild isolates of a panel of SARS-CoV-2 lineages, including B.1, Zeta, N.10, and the VOCs Gamma, Alpha, and Delta that emerged in different timeframes of the pandemic. The main objectives of the present study were to evaluate if serum of COVID-19 patients infected by earlier lineages of SARS-CoV-2 were capable to neutralize recently emerged VOCs, and if PRNT90 is a reliable serologic method to distinguish infections caused by different SARS-CoV-2 lineages. Overall, sera collected from the day of admittance to the hospital to 21 days after diagnostic of patients infected by the two earlier lineages B.1.1.28 and B.1.1.33 presented neutralizing capacity for all challenged VOCs, including Gamma and Delta, that were the most prevalent VOCs in Brazil. Among all variants tested, Delta and N.10 presented the lowest mean of neutralizing antibody titers, and B.1.1.7, presented the highest titers. Four patients infected with Gamma, that emerged in December 2020, presented neutralizing antibodies for B.1, B.1.1.33 and B.1.1.28, its ancestor lineage. All of them had neutralizing antibodies under the level of detection for the VOC Delta. Interestingly, patients infected by B.1.1.28 presented very similar mean of neutralizing antibody titers for both B.1.1.33 and B.1.1.28. Findings presented here indicate that most patients infected in early stages of COVID-19 pandemic presented neutralizing antibodies up to 21 days after diagnostic capable to neutralize wild types of all recently emerged VOCs in Brazil, and that the PRNT90 it is not a reliable serologic method to distinguish natural infections caused by different SARS-CoV-2 lineages.

understood, some studies have confirmed neutralizing antibodies as an immune correlate of 47 protection [2] . The humoral immune response can block infection through neutralizing 48 antibodies, which bind the virus in a manner that prevents host cell infection [3] . The host 49 humoral response against SARS-CoV-2, including IgA, IgM, and IgG response, has been 50 examined mostly by ELISA-based assays using recombinant viral nucleocapsid protein or 51

pseudovirus-based neutralization assays [4] . Coronavirus infections typically induce neutralizing 52 antibody responses, and virus neutralization assays performed on cell cultures, as plaque 53 reduction neutralization test (PRNT), are considered as gold standard for serological testing and 54 determining immune protection [1] . Although antiviral T and B cell memory certainly contribute 55 some degree of protection, strong evidence of a protective role for neutralizing serum antibodies 56

exists [5] . Neutralizing antibody levels are highly predictive of immune protection from 57 symptomatic SARS-CoV-2 infection [2] . 58 Since the pandemic began in China in December 2019, thousands of SARS-CoV-2 59

lineages have emerged worldwide [6] . The variants that presented increased transmissibility, 60 virulence, and decreased response to available diagnostics, vaccines, and therapeutics were 61 defined by the World Health Organization as variant of concern (VOC) [7] . Following the 62 upsurge of variants, several reinfection cases started to be reported worldwide rising questions 63 about the efficiency of humoral response mounted after primary infections to prevent a 64 secondary infection by SARS-CoV-2 [8] . Elmer, www.chemagen.com). SARS-CoV-2 was detected by qRT-PCR assays targeting the viral 92 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. for Delta in all individuals (Table) . 164 165 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. (Alpha, Gamma, and Delta). 174 175 176

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. *NT: Not tested; for geometric mean calculation purposes, 1 was used for titers <10, and <20.

.

It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. 

The relationship between humoral response to SARS-CoV-2 antigens, especially the spike protein, and clinical protection from COVID-19 remains not fully understood, although some studies have confirmed neutralizing antibodies as an immune correlate of protection [2] . Likewise, it is still not completely understood to what extent the mutations in viral antigens contribute to virus evasion from neutralizing antibodies, and how significant this escape mechanism could be for the general effectiveness of the protective response, especially in the context of vaccinations, reinfections, and the evolution of the pandemic. It has been shown that mutations in the ACE2 binding site of variants result in an increased affinity for the receptor ACE2, and that changes outside the receptor-binding domain also impact neutralization [17] . In this scenario, we primarily investigated the neutralization profile of sera from patients infected by the most prevalent lineages in early pandemic in Brazil including B.1.1.33 and B.1.1.28, with the recently emerged VOCs including Gamma, Alpha, and Delta. Additionally, we investigated if Gamma-induced antibodies were capable to neutralize the early isolates and the most recently emerged VOC, Delta. We observed that, despite the variation in antibody levels, most serum samples of patients infected by early isolates presented some level of neutralizing activity against all VOCs. The reverse situation, in which serum samples from individuals infected with the Gamma lineage were challenged with early pandemic viral isolates, also produced detectable neutralization. However, when sera from Gamma-infected individuals were tested with other VOC Delta, the PRNT 90 titer was below the limit of detection of the assay (Table) .

Gamma, which emerged from the B.1.1.28 lineage, contains 17 amino acid substitutions, ten of which in the spike protein, including N501Y, E484K and K417T in the receptor-binding domain, five in the N-terminal domain, and the mutation H655Y near the furin cleavage site [18] . These mutations reduce the neutralization capacity of convalescent sera from individuals infected by early isolates [11] , and findings presented here suggest these mutations perhaps interfere also with the efficiency of its neutralizing antibodies for other variants, as Delta.

Despite the small number of patients evaluated, the findings that Gamma-induced immune sera presented low or absent PRNT 90 titers for Delta raises concern over the potential risk of reinfections, and consequent prolongation of the pandemic. Gamma was the most prevalent lineage of SARS-CoV-2 in Brazil for several months in a row, and now, people who were previously infected by Gamma are being exposed to Delta, which is currently the most prevalent VOC in the country [16] . In Brazil, Delta was first detected in May 2021, and by the end of August, 70% of the genomes of SARS-CoV-2 sequenced were Delta (www.genomahcov.fiocruz.br/dashboard/). As previously reported, this variant presents a higher replication fitness and decreased sensitivity to neutralizing antibodies mounted for early isolates, both contributing to higher transmissibility [19] .

It is important to mention that the protective role of neutralizing antibodies does not seem to be directly linked to the titers in which they are found, but to timing and kinetics of their production, with studies suggesting a limited role of antibodies in predicting disease severity of the COVID-19, and that the earlier the presence of neutralizing antibodies after infection, the less severe is the disease outcome [20] .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.24.22269379 doi: medRxiv preprint It is noteworthy that for most viruses there is no direct correlate of protection in humans, since the studies needed to establish such a correlate in humans are challenging. For instance, protective neutralizing antibody titers have been roughly estimated for yellow fever vaccine by challenge studies in nonhuman primates and hamster models [21, 22] . The threshold of protective neutralizing antibody titers for SARS-CoV-2 and its lineages has not been established. Therefore, lower neutralizing antibodies titers including the samples with titers under the limit of detection as <10, do not necessarily mean susceptibility to a hypothetical secondary challenge. Samples that presented titers below 10 were not tested in lower dilutions, and if titers ≤ 9 are protective for each one of the different lineages of SARS-CoV-2 tested remain unknown.

Despite concerns over the potential risk of infections caused by Delta variant in patients previously exposed to Gamma, it is noteworthy that the number of reinfections reported worldwide remains limited, and that reinfections caused by Delta variant after previous infection by another VOC, as Gamma, have not been reported yet. The continuous advance of the COVID-19 vaccination has reduced the pace of new infections worldwide and is also expected to mitigate the number of reinfections as well. It is important to mention though that the time Delta variant became dominant in Brazil coincides with the local increasing vaccination coverage, but because of the unequal pace of vaccination throughout the country and along the time, Brazil remains with vaccination coverage below desirable. By the beginning of October 2021, over 58% of the adult population had been fully vaccinated in Brazil [23] . Regardless the recent improvement, a recent study conducted in the United States found that if in the state of Florida, the coverage was 74% instead of 59,5%, vaccination would have averted 664,007 additional cases, and reduced hospital admissions by 61,327, and deaths by 16,235 [24] .

Finally, cellular immune response is believed to play also an important role in the immune response [5] . Cellular immune response was not investigated in the present study, and only with a combined evaluation of both immune responses for a better understanding of the relationship between neutralizing antibody titers and susceptibility to secondary infections.

Besides being considered markers of immune protection, specific neutralizing antibodies have also been used to evaluate viral exposure, and ultimately, for diagnostic purposes. Assays designed to detect neutralization antibodies have been widely used for the diagnostic of different viral groups. The PRNT is the most specific and gold standard serological test for the differentiation of closely related flavivirus infections, as dengue and yellow fever viruses in convalescent serum samples [25] . Type-specific antibodies can be distinguished using the PRNT, and two or more flaviviruses are distinct from each other by quantitative serological criteria. Four-fold or greater differences between PRNT titers have been used as seropositivity criterion in heterologous reactions [25, 26] . In the present study, despite some genetic and immunogenic differences observed among all lineages of SARS-CoV-2 worldwide, these viruses are closely related and for that reason a PRNT with a highly conservative threshold of 90% neutralization in ≥ 1:10 serum dilution was used as seropositivity criterion.

According to our findings, the four-fold or greater difference among PRNT titers was not a suitable seropositivity criterion to distinguish infections among the different lineages of SARS-CoV-2 tested. Convalescent serum from patients infected by B.1.1.28 presented a mean of PRNT titer very similar for B.1.1.28, B.1.1.33 and Alpha lineages, and the titer difference was less than four-fold greater than titers for B.1. Four-fold or greater titers for B.1.1.28 were observed only when compared to the titers of the variants N.10, Zeta and Delta.

The similar profile was observed for samples from patients infected by B.1.1.33. In this case, these patients presented a non-significant difference in PRNT titers between B.1.1.33, B.1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.24.22269379 doi: medRxiv preprint and Alpha. B.1.1.33-induced sera presented a mean of PRNT titer less than four-fold greater for B.1.1.33 when compared to B.1.1.28, Zeta and N.10. Four-fold or greater B.1.1.33 titers were observed only when compared to Gamma and Delta VOCs. Finally, for the patients previously infected by Gamma, PRNT titers for Gamma were more than four-fold higher for Gamma when compared to B.1 and B.1.1.28, Delta and Zeta. Differences were smaller than four-fold when compared to B.1.1.33, N.10, and Alpha. Of note, the mean of PRNT 90 titers for the Alpha variant was the second highest in all the three groups of patients, which includes samples of patients infected by B.1.1.28 and B.1.1.33 that were collected before the upsurge of Alpha variant in the United Kingdom.

The difference in PRNT 90 titers demonstrated here among all lineages indicate that the four-fold or greater criterion of seropositivity is not reliable to distinguish lineage infections. The same is truth if considering optionally two-fold or greater differences as criterion de seropositivity.

In conclusion, the humoral response mounted for more recently emerged variants after natural infections caused by earlier isolates and different lineages reported here, corroborates the importance of the international recommendation of a high vaccination coverage which has being instrumental to reduce severe cases and deaths, mitigate virus propagation, and the upsurge of new variants.

Author Correction: A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

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We gratefully acknowledge the patients and authors from the originating laboratories responsible for obtaining the specimens. We thank the funding support from General Laboratories .