Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine | bioRxiv

Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine | bioRxiv

Key Points

This paper investigates the neutralizing antibody responses against different SARS-CoV-2 variants after vaccination with two doses of the Pfizer-BioNTech COVID-19 vaccine. Here are the key findings:

• Serum samples were collected from 14 participants, including 12 vaccinated with two doses of Pfizer's vaccine, median 21 days after the second dose. The sera were tested against three SARS-CoV-2 strains - wildtype, Beta variant, and Delta variant - using a live virus neutralization assay.
• Neutralizing antibody titers (nAbT) were significantly lower against Delta variant compared to wildtype, with a 4.4-fold reduction. nAbT against Beta variant were also reduced 2.3-fold versus wildtype.
• No neutralizing antibodies were detected in the two unvaccinated participants, nor in one immunosuppressed vaccinated participant. The highest nAbT was seen in a participant infected with SARS-CoV-2 prior to vaccination.
• Sequencing of viruses after neutralization revealed limited consensus-level mutations compared to inoculum viruses. However, numerous minority variant alleles (MVAs) were detected, especially in the spike protein of all variants.
• One spike MVA resulting in mutation D1258H was detected in 41/96 infections post-neutralization, but not in the inoculating viruses, suggesting a potential culture adaptation.

In summary, there is significantly reduced neutralization of Beta and Delta variants versus wildtype SARS-CoV-2 after two Pfizer vaccine doses. Ongoing viral genomic surveillance during neutralization assays is warranted to monitor for novel mutations arising in culture.

Utility in Developing Improved Vaccines

Studies like this that investigate viral evolution and antibody neutralization against variants can help guide the development of improved booster vaccines. Here are some key ways these types of studies could aid next-generation vaccine design:

1. Identify mutations that enable immune evasion: By testing serum from vaccinated individuals against different variants, researchers can pinpoint specific mutations that reduce neutralization. These sites can be targeted in updated vaccines.
2. Monitor for new variants of concern: Continued surveillance during neutralization assays can detect new mutations arising in vitro that may become future variants of concern in vivo. This early warning could facilitate variant-specific vaccines.
3. Determine need for variant-specific boosters: If certain variants show markedly reduced neutralization, it provides evidence that variant-specific vaccine boosters may be warranted to restore protection.
4. Prioritize variants for boosters: By testing a panel of variants, researchers can determine which ones show the greatest immune evasion and should be prioritized for variant-specific boosters.
5. Evaluate breadth of protection: Testing against multiple variants reveals whether vaccine responses are broadly protective or if they are strain-specific. This supports designing broad boosters.

So in summary, viral genomic studies enable monitoring of immune evasion mutations and variants. These data help guide the design of next-generation mRNA and other vaccines to optimize protection against emerging variants of concern.

 Viral genomic surveillance

Viral genomic surveillance during neutralization assays typically involves sequencing viruses before and after neutralization selection pressure to look for genomic changes. The key steps are:

Sample Collection:

• Inoculum viruses and post-neutralization samples are collected for sequencing. Multiple biological replicates are tested.

Nucleic Acid Extraction:

• Viral RNA is extracted from the samples using viral nucleic acid extraction kits.

Enrichment and Library Preparation:

• The viral RNA undergoes target enrichment using probes or PCR to amplify viral sequences over host/microbial background.
• Enriched viral cDNA is prepared into sequencing libraries with barcodes for each sample.

Sequencing:

• Libraries are sequenced using next generation platforms like Illumina to get high coverage reads.

Bioinformatics Analysis:

• Raw reads are quality filtered, aligned to a reference genome, and variants are called to identify consensus mutations and minority alleles.

Key instruments and tools include:

• Biosafety infrastructure for viral culture and handling (BSL-3 lab)
• Nucleic acid extraction systems (columns, magnetic beads)
• Target enrichment systems like the Respiratory Viral Oligo Panel (RVP)
• Next generation sequencers like Illumina MiniSeq or MiSeq
• Bioinformatics software for genome assembly, alignment, and variant calling.

Overall this allows high resolution tracking of viral evolution during in vitro selection experiments.

The viral genomic surveillance approach described in this paper was applied to viruses generated in cell culture, not directly on samples from COVID-19 patients.

The inoculum viruses used were originally derived from symptomatic COVID-19 patients - the authors state they used respiratory samples that tested positive for SARS-CoV-2. However, they then cultured the viruses in Vero E6 cells to generate stocks for neutralization experiments.

After neutralization selection with vaccinated sera, samples were taken for sequencing to look for viral evolution. So the sequencing was done on the cultured viruses, not the original clinical specimens.

Direct sequencing of viruses in COVID-19 patient samples can be more challenging due to:

• Lower viral titers compared to culture
• Presence of host genomic DNA background
• Need to sequence directly without culture steps

Some key differences for patient sample sequencing:

• Requires high viral titers so there is enough viral RNA for sequencing
• Uses techniques like multiplex PCR or target enrichment to focus sequencing on viral RNA over host/microbiome
• Bioinformatics needs to account for sequencing errors, contaminants, and lower viral coverage

This paper used cultured virus stocks derived from COVID-19 patients, rather than direct clinical samples, for the neutralization selection and viral sequencing experiments. But patient samples are sequenced in other genomic surveillance studies.

Neutralizing antibody titers (nAbT)

Neutralizing antibody titers (nAbT) refer to the measurement of levels of antibodies in serum that are capable of neutralizing a virus. It is a quantitative assay used to evaluate the potency of antibodies against a viral infection.

The neutralization assay works by mixing serial dilutions of serum samples with a fixed amount of virus and then adding the mixture to cells. The nAbT is determined by identifying the highest serum dilution (titer) that neutralizes the virus and prevents infection of the cells.

Some key points about neutralizing antibody titers:

• They provide a quantitative estimate of the levels of functional, virus-neutralizing antibodies in serum.
• Higher nAbT indicates greater concentration of neutralizing antibodies. A high titer confers more potent antiviral immunity.
• Testing against different viral strains can evaluate cross-neutralization potency against variants.
• Used to assess protective immunity from vaccines or natural infection. High nAbT correlated with protection.
• Declining nAbT over time can indicate waning immunity. This helps guide booster schedules.
• Limited by the assay conditions. Results can vary depending on cell types, viral dose, endpoints measured.

In the context of this study, the nAbT results help compare the neutralizing potency of serum antibodies against the wildtype, Beta, and Delta SARS-CoV-2 variants. The reductions in nAbT against the variants indicates decreased neutralization activity.

Key Question

The key question answered in this paper is whether two doses of the Pfizer-BioNTech (BNT162b2) COVID-19 vaccine are as effective at neutralizing the Delta variant compared to the Beta variant and wildtype SARS-CoV-2. The main findings are:

• There was a significant 4.4-fold reduction in neutralizing antibody titers against the Delta variant compared to wildtype.
• There was also a 2.3-fold reduction against Beta variant versus wildtype.
• The sera still neutralized the variants, but at lower levels compared to wildtype.

So in summary, yes the data indicates the vaccine is less effective against Delta and Beta variants compared to the original wildtype virus.

However, the study has some limitations:

• It only looked at samples collected a median of 21 days after the second dose. No data on duration of immunity over months.
• Small sample size of 14 participants.
• Looked only at antibody neutralization, not full immune response.

So while it suggests reduced efficacy against variants, particularly Delta, it does not provide full data on long-term immunity or necessity of boosters. The reductions were based on lab neutralization assays, and further data is needed to correlate with real-world effectiveness against infection and severe disease. Additional clinical studies over longer periods can better address duration of protection and need for boosting.