virology blog

About viruses and viral disease

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Heterologous Vaccine Regimens Might be Better

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by Gertrud U. Rey

Have you ever wondered if you can “mix and match” SARS-CoV-2 vaccines? For example, would it be ok to boost a first dose of the vaccine produced by AstraZeneca with a dose of the vaccine produced by Pfizer/BioNTech? The latest science shows that such a vaccine regimen actually induces a stronger immune response than a regimen consisting of two doses of the same vaccine.

The occasional incidence of thrombosis in people under the age of 60 after receiving an adenovirus-vectored vaccine like the ones made by AstraZeneca and Johnson & Johnson has prompted several European governments to recommend the use of these vaccines only in people over 60. Because many people under 60 had already been vaccinated with a first dose of the AstraZeneca vaccine and still needed a second dose, they had to decide whether they should continue their regimen with another dose of the same vaccine (i.e., a homologous regimen), or receive an mRNA vaccine instead (i.e., a heterologous regimen).

In an effort to evaluate the efficacy of a heterologous SARS-CoV-2 vaccine regimen, the authors of this Brief Communication engaged the participants of an ongoing clinical trial, which aims to monitor the immune responses to SARS-CoV-2 in health care professionals and individuals with potential exposure to SARS-CoV-2. All study participants had already received a first dose of the AstraZeneca vaccine (referred to as “ChAd” in the study) and were given the option of receiving a second dose of the same vaccine or a dose of Pfizer/BioNTech’s mRNA vaccine (referred to as “BNT”). Although both of these vaccines encode the gene for the full-length SARS-CoV-2 spike protein, it is housed slightly differently. In the ChAd vaccine, the spike protein is encoded in an adenovirus vector of chimpanzee origin, while in the BNT vaccine it is surrounded by a lipid nanoparticle.

Out of the 87 individuals who participated in the study, 32 chose a second dose of ChAd and 55 chose to be vaccinated with BNT instead. Participants who chose a homologous ChAd/ChAd regimen received their second dose of ChAd on day 73 after the initial dose and donated a blood sample for analysis 16 days later. Participants who chose a heterologous ChAd/BNT regimen received their dose of BNT 74 days after their initial ChAd dose and donated a blood sample 17 days later. All results obtained from the analyses of the blood samples from these two groups were compared to results obtained from a group of 46 health care professionals who had been vaccinated with two doses of BNT (i.e., a homologous BNT/BNT regimen).

Briefly, the findings were as follows. Relative to the antibody levels induced by the first dose, homologous boosting with ChAd led to a 2.9-fold increase in IgG antibodies against the SARS-CoV-2 spike protein. IgG antibodies are mostly present in the blood and provide the majority of antibody-based immunity against invading pathogens. In contrast, heterologous boosting with BNT led to an 11.5-fold increase in anti-spike IgG, within the range observed in BNT/BNT-vaccinated individuals. A similar pattern was observed with anti-spike IgA antibodies, which are predominantly found in mucus membranes and their fluids. Boosting with BNT induced significantly higher increases in anti-spike IgA than did boosting with ChAd, suggesting that a heterologous boosting regimen induces better antibody responses. Interestingly, although the booster immunization induced an increase in neutralizing (i.e., virus-inactivating) antibodies in both vaccination groups, only heterologous ChAd/BNT vaccination induced antibodies that neutralized all three tested variants (Alpha, Beta, and Gamma), similar to what was observed in BNT/BNT vaccine recipients.

The authors also analyzed the effect of the two different boosting regimens on spike-specific memory B cells, which circulate quiescently in the blood stream and can quickly produce spike-specific antibodies upon a subsequent exposure to SARS-CoV-2. All vaccinees from both the ChAd/ChAd and ChAd/BNT groups produced increased levels of spike-specific memory B cells after receiving their booster shot, with no significant differences observed between the two groups. These results emphasize the importance of booster vaccination for full protection from SARS-CoV-2 infection.

ChAd/BNT recipients also had significantly higher levels of spike-specific CD4+– and CD8+ T cells compared to ChAd/ChAd recipients. CD4+ T cells are a central element of the adaptive immune response, because they activate both antibody-secreting B cells and CD8+ T cells that kill infected target cells. Compared to the ChAd/ChAd regimen, ChAd/BNT vaccination also induced significantly increased levels of T cells that produce spike-specific interferon gamma – a cytokine that inhibits viral replication and activates macrophages, which engulf and digest pathogens. Overall, these results suggest that a heterologous ChAd/BNT regimen induces significantly stronger immune responses than a homologous ChAd/ChAd regimen.

The study did have several limitations. First, it was not a randomized, placebo-controlled trial where each participant was randomly assigned to an experimental group or a control group. Randomizing trial participants eliminates unwanted effects that have nothing to do with the variables being analyzed, so that the only expected differences between the experimental and control groups are the outcome variable studied (efficacy in this case). Subjects in a control group receive a placebo – a substance that has no therapeutic effect, so that one can be sure that the effects observed in the experimental group are real and result from the experimental drug. Second, the authors were unable to test the antibodies for their ability to neutralize the Delta variant, which has recently become the predominantly circulating variant in many parts of the world. Third, the study mostly included young and healthy people, making it difficult to extrapolate the data to other specific patient groups outside of this category. Fourth, the data only show the results of assays performed in vitro, which may not necessarily manifest a clinical significance. Extended studies aimed at determining the practical importance of the observed immune responses are needed to validate the relevance of these responses. 

Despite its limitations, the study provides information that could have some valuable practical implications. Most of the currently approved SARS-CoV-2 vaccine regimens involve two doses of the same vaccine. However, access to two doses of the same vaccine may be limited or absent under some circumstances, thus necessitating the use of a different type of vaccine to boost the first dose.

[For a more in-depth discussion of this study, I recommend TWiV 782.]

Holiday travel explains spread of a SARS-CoV-2 variant

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coronavirus Spike

The emergence and spread throughout Europe of a SARS-CoV-2 variant, 20E (EU1) in the summer of 2020 illustrates how a virus may become dominant not by increased transmissibility but through travel and lack of effective containment and screening.

The SARS-CoV-2 variant 20E (EU1) emerged in Spain in the summer of 2020 and spread to multiple European countries. By the fall of 2020 most of the sequences in Europe were from 20E (EU1). The variant bears a number of amino acid changes including A222V in the N-terminal domain of the spike protein (pictured).

Results of binding and neutralization assays revealed that the A222V change did not affect interaction of the spike protein with polyclonal or monoclonal antibodies. Lentivirus particles bearing the spike with A222V did not reproduce with higher efficiency in cells in culture. Authors conclude that these observations are not consistent with increased transmissibility of the 20E (EU1) variant. However, I would argue that studying the infectivity of a pseudotyped virus – in this case a lentivirus bearing the SARS-CoV-2 spike – in 293 cells (possibly human embryonic kidney) has virtually no relevance to what occurs in humans. At the very least, authentic SARS-CoV-2 infection of human respiratory tract cells must be examined. Even then, the results may have no direct bearing on what occurs in humans.

In contrast, epidemiological evidence explains the spread of 20E (EU1). This variant arose first in Spain in early summer 2020. It then spread extensively in Spain and also spread to other European countries. Results of modeling indicate that the spread of the variant can be explained by holiday travel-associated transmission (many EU countries opened their borders to travel on 15 June), and human behaviors such as failure to distance, mask, restrict gatherings, and adequately test for infections.

It seems likely that human behavior might also account for the global spread of other SARS-CoV-2 variants (e.g. alpha and beta). Authors believe that such spread is due to increased inherent transmission of the viruses, but the data in support of this conclusion are not convincing. Increased reproduction of pseudotyped viruses in cells in culture, as pointed out above, is likely irrelevant. Increased shedding of viral RNA from the nasopharynx, detected by PCR, is also irrelevant as it does not represent infectious virus. In no case has shedding of infectious virus from the nasopharyngeal tract been studied to address potential mechanisms of increased transmission. It is claimed that the reproductive index of variants is increased, but these calculations are flawed. The reproductive index is determined by a formula that includes both viral and host factors. However, host factors are never included when this index is determined for individual variants. As the study above indicates, human activities can substantially affect predominance of a virus in a population.

Why do variants out-compete and displace other viruses? It is because the variants have increased fitness, the ability of a virus to reproduce in the host. Fitness can be altered in many ways, including evasion of antibody responses, increased particle stability, and even person to person transmission. No experiments have been done to explain the increased fitness of variants. Fitness is not the same as transmission.

Variants of influenza virus arise frequently, and these variants displace existing viruses because they have a fitness advantage. For influenza virus, a fitness advantage is often conferred by HA amino acid changes that allow escape from antibody neutralization. Antigenic variants can infect a slightly larger number of hosts and that is enough natural selection advantage for the new variants to outcompete the older ones. No one ever says that these influenza virus variants have increased transmission.

Unfortunately the narrative that the variants have increased transmission is dominating the media. This situation has arisen because virologists, epidemiologists, and evolutionary biologists are not talking to one another. In addition, what we know about other viruses, as illustrated here for influenza virus, is also ignored.

SARS-CoV-2 variants arise during individual infections

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Early in the COVID-19 pandemic, SARS-CoV-2 genome variation appeared to be low, with an average of 10 base differences in the 30,000 base genome between any two isolates. Late in 2020, as many more people were infected, variants were isolated that had more changes than previously seen. A study of variation at the genome level indicates that diversity in a single host has been underestimated.

SARS-CoV-2 variants certainly arise in each infected individual, and these may be subjected to selection pressures such as those imposed by antibodies binding to spike protein. Commonly used sequencing technologies do not permit detection of virus variants within single samples. To address this limitation, a high-throughput sequencing method was developed for sequencing individual virus RNA genomes in patient samples. The results demonstrate that SARS-CoV-2 variants emerge in each host and are subject to immune selection pressure.

When this method was used to analyze a patient isolate of SARS-CoV-2 after four passages in Vero cells, in the regions encoding spike, ORF3, envelope, and M protein, 18 unique combinations of mutations were detected. Over half of the single genome sequences differed from the reference sequence. Many of the changes led to amino acid substitutions, suggesting selection pressure occurring during cell culture adaptation.

In-host diversity was then examined for 7 patients sampled between days 8 and 17 of illness onset. Only a few sequence haplotypes were detected, with no clear consensus among the amino acid changes. These relatively homogeneous sequences might have been a consequence of sampling early in infection when limited antibody pressure was present. Examination of the antibody response in longitudinal samples revealed an increasing and broadening antibody response to the spike.

A study of antibody responses and presence of mutations in individual genomes revealed a correlation between the emergence of antibodies to the N-terminal domain of spike protein, the emergence of amino acid changes in regions that bind these antibodies, and a delay in clearance of virus. These results are consistent with antibody pressure during infection leading to selection of SARS-CoV-2 spike variants (pictured).

SARS-CoV-2 variation in a single host had not been previously described, probably due to the inability of previous sequencing methods to distinguish variation from technical errors. It is also likely that high-quality data were acquired early in infection when viral titers are high yet antibody pressure is not yet present.

There are several take-home messages from these findings. They illustrate how diversity in a single host might give rise to antibody-resistant variants that could spread in a population if they have the appropriate fitness. In-host escape might be avoided by containing viral reproduction early in infection, either by prior immunity or by antiviral therapy, before selection by antibody takes place. This method should be applied to studying more COVID-19 patient samples to understand the extent of potential in-host SARS-CoV-2 variation.

Understanding virus isolates, variants, and strains

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Many virology terms are being used these days by people who do not understand their meaning. Included are journalists, medical doctors, scientists, lawyers, and people from all walks of life. In normal times this word mis-usage would be so rare that it would not matter. However, because we are in a viral pandemic that affects nearly everyone, I will attempt to explain the meaning of virus isolates, variants, and strains.

Many of the terms used in virology are ill-defined. They have no universally accepted definitions and there is no ‘bible’ with the correct meanings. As each of us are trained by other virologists, we hear them using terms in certain contexts and we copy their usage – whether or not it is correct. I learned many good things from my mentors but also many things that are wrong.

Nevertheless, certain terms should have specific meanings. Some of my colleagues will certainly disagree with some of my definitions, others will agree. Kudos to the latter. I also recognize that few will read this post and it will have little impact. Perhaps one day a high school student will search for some of the terms and come across it. It is mainly meant for me to put my thoughts down in an orderly manner.

The virology terms I have in mind all have to do with attempts to place order on the huge varieties of viruses in the virosphere. Most of them today derive their meaning from the viral genome: the DNA or RNA that encodes the production of new virus particles. This reliance on the genome is relatively recent: until the 1980s we had no genome sequences; hence most categories were based on other properties, such as the size of the virus particle, whether or not it has a membrane, its type of symmetry, and much more. Today it’s all about the genome. Whether or not you think this myopia is a good idea is not the topic of this post.

Let’s start with the term virus isolate, because it’s the easiest to define. An isolate is the name for a virus that we have isolated from an infected host and propagated in culture. The first isolates of SARS-CoV-2 were obtained from patients with pnemonia in Wuhan in late 2019. A small amount of fluid was inserted into their lungs, withdrawn, and placed on cells in culture. The virus in the fluid reproduced in the cells and voila, we had the first isolates of the virus.

Virus isolate is a very basic term that implies nothing except that the virus was isolated from an infected host. An isolate comes from a single host. We can have my virus isolate, or yours, or the neighbor’s down the street. Most patients do not get to have virus isolates taken from them. Even though SARS-CoV-2 has infected millions, we do not have millions of isolates, probably just thousands. We do have genome sequences from many people, and those can be inferred to represent the isolate from each person – however in most cases infectious virus is not isolated from individual patients.

Isolates are given names so that their origin is known. For example, one of the early isolates of SARS-CoV-2 is called BetaCoV/Wuhan/WIV04/2019. This isolate name consists of the genus, Betacoronavirus, followed by the city of origin, the isolate number, and the year. SARS-CoV-2 is the name of the virus; it is not an isolate name. Isolates of other viruses are also precisely named. I’m a big fan of the very detailed influenza virus nomenclature, which is as follows: Virus name/antigenic type/host of origin if other than human/geographical origin/serial number/last two digits (or all four digits) of year of isolation/hemagglutinin subtype neuraminidase subtype. Examples include influenza A virus A/duck/Germany/1868/68 (H6N1) or influenza A virus A/chicken/Vietnam/NCVD- 404/2010 (H5N1).

A virus variant is an isolate whose genome sequence differs from that of a reference virus. No inference is made about whether the change in genome sequence causes any change in the phenotype of the virus. The meaning of variant has become clouded in the era of whole viral genome sequencing, because nearly every isolate may have a slightly different genome sequence. Such is the case for SARS-CoV-2: nearly every sequence from a different person is slightly different. Up until the end of 2020, any SARS-CoV-2 sequences from any two individuals differed by about ten nucleotide changes out of 30,000. They are all variants, but the term is rarely used in this context. However since then viral genomes with many more changes have been identified. These have been called ‘variants of concern’ (VOC) because it is thought that the changes confer new phenotypic properties such as increased fitness. British scientists did a good deed by calling them VOCs, because now the press must call them variants.

Unfortunately mainstream media, following in the footsteps of scientists who really should know better, have been using the term ‘strain’ to describe what are actually variants. This practice emerges in every viral outbreak: there is a new, more (fill in the blank with your favorite phenotype) strain of Ebolavirus, of Zika virus, and now of SARS-CoV-2. It began early in 2020 with the finding of variants with a single amino acid change in the spike protein, from D to G at position 614. The press called this a new strain that was more transmissible. But the use of strain was incorrect: it is a variant and remains so to this day.

A virus strain is a variant that possesses unique and stable phenotypic characteristics. Such characteristics can only be ascertained by the results of experiments done in the laboratory, in cells in culture and in animals, coupled with observations made in infected humans. The name strain is not easily earned: certainly it cannot simply be given by journalists! As Jens Kuhn has written, “The designation of a virus variant as a strain would be the responsibility of international expert groups”. No such designation of strain has been given more than once to SARS-CoV-2: there is one, and only one strain of this virus. No incorrect usage of that term will change this fact. As you might imagine, it can take some time for an international group of experts to agree on anything.

Viral strains are few and far between: it is a designation highly desired but given sparingly. A retrovirologist recently assured me that there is only one strain of HIV-1. The Lansing strain of poliovirus is derived from a human isolate that was passaged 99 times in mice until it acquired the ability to infect that species. That strain has demonstrably different properties from the human strain.

There are other terms to describe viruses but they are more confusing than contentious, and they are not used universally. The term serotype is used to describe viruses of the same species that are antigenically different. There are three serotypes of poliovirus; if you are infected with type 1, then immunity you generate will not protect you against infection with types 2 or 3. Same for the four serotypes of dengue virus, and the hundreds of rhinovirus serotypes. These days, the genome sequence of the virus is used to infer whether isolates are serologically different. The term genotype is used to describe the genetic makeup of a virus. For example, hepatitis C viruses are placed in different genotypes depending on the overall identity of their genomes. For other viruses, the term clade is used. A clade is a group of organisms composed of an ancestor and its descendants, as illustrated by the phylogenetic tree below. SARS-CoV-2 isolates and HIV-1 isolates are placed in clades based on phylogenetic trees constructed from their genome sequences.

I believe that the terms of virology should be used accurately and consistently. The terms isolate, strain, and variant have been frequently and incorrectly misused during the pandemic, which generates confusion. I have little faith that either the general public or the scientists will agree on any nomenclature. Rest assured that if you misuse isolate, variant, or strain, I will correct you according to my lexicon.