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About viruses and viral disease

Vincent Racaniello

Understanding virus isolates, variants, and strains

25 February 2021 by Vincent Racaniello 7 Comments

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 A small amount of fluid was inserted into their lungs, withdrawn, and placed on cells in culture.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.

SARS-like bat coronaviruses are not only in China

18 February 2021 by Vincent Racaniello 7 Comments

It is well past the time to stop blaming a laboratory in China for the release of SARS-CoV-2. Such fallacies reflect an ignorance of scientific facts, including the recent finding of closely related coronaviruses in bats in Thailand.

The bat CoV RatG13, sampled in 2013 in Yunnan province, shares 96% whole genome identity with SARS-CoV-2, suggesting a likely bat origin of the pandemic virus. To identify other possible sources for highly related viruses, a colony of 300 bats in eastern Thailand, consisting only of one species, Rhinolophus acuminatus, was sampled in June 2020. Thirteen of 100 bat rectal swab samples were positive for a single PCR amplicon with 95.86% sequence identity to SARS-CoV-2 and 96.21% identity to bat CoV-RaTG13. This virus, named RacCS203, appears to be the dominant coronavirus circulating in this bat colony. Phylogenetic analyses indicate that RacCS203 is a new member of the SARS-CoV-2 related CoV lineage (SC2r-CoV).

[Read more…] about SARS-like bat coronaviruses are not only in China

An antiviral to prevent or treat SARS-CoV-2 infection

11 February 2021 by Vincent Racaniello 4 Comments

Vaccine development has far outpaced antiviral discovery for COVID-19. Hydroxychloroquine was a disaster, and the repurposed remdesivir, which must be administered intravenously, has modest effect when given to hospitalized patients. The situation is unfortunate because antiviral drugs may be used to either prevent infection (prophylactic) or treat infection (therapeutic). A promising antiviral drug candidate is EIDD-2801/MK-4482 which has been shown to block SARS-CoV-2 transmission among ferrets, and more recently, to treat or prevent infection in mice. In both cases the drug is active after oral administration.

[Read more…] about An antiviral to prevent or treat SARS-CoV-2 infection

SARS-CoV-2 variants of concern

28 January 2021 by Vincent Racaniello

In recent months variants of SARS-CoV-2 have been detected that are unusual in that they have many more genome mutations than previously found. These have been called ‘variants of concern’ (VOC) as it has been suggested that the genome mutations might impact transmission, immune control, and virulence. Below I cover each of these issues separately.

Transmission

The SARS-CoV-2 lineage called B.1.1.7 arose in the United Kingdom in September 2020 and harbors 17 genomic mutations, some of which lead to amino acid changes in the spike protein (pictured). Similar but distinct variants have been detected in other locations, including South Africa (B.1.135) and Brazil, but the B.1.1.7 lineage has been best studied. A good summary of the changes can be found in this manuscript. A number of lines of evidence have led to the conclusion that viruses of the B.1.1.7 lineage may have increased transmissibility compared with previous isolates. These include the rapid displacement of previous variants in the UK within a short period of time; an apparent increase in the R index for such variants; and increased levels of viral RNA in nasopharyngeal washes as measured by PCR or RNA sequencing.

The virological definition of transmission is the movement of viruses from one host to another. In the case of SARS-CoV-2, such transmission occurs when infectious virus particles are exhaled within respiratory droplets and arrive in another host, where they initiate infection. The evidence cited above for increased transmission of the B.1.1.7 lineage are all indirect and do not prove that the variants actually transmit, in a virological sense, better between hosts. The population growth of the variant could, for example, be a consequence of changes in human behavior. The R index, a measure of transmisssibility, is influenced not only by the virus but by human behavior. The finding of increased levels of RNA in nasopharyngeal wash is also inconclusive with respect to transmission. Viral RNA is not the same as infectious virus, and no studies have been done measuring shedding of infectious virus from individuals infected with variants of the B.1.1.7 lineage compared with other variants.

There is no doubt that the B.1.1.7 lineage has rapidly displaced others in the UK. Whether this behavior is due to an increased ability of the virus to be transmitted form one host to another has not been demonstrated. The variant has also been detected in other countries and its dispersion in those locations are not consistent with increased transmission (as I have defined above). For example, we now know that the B.1.1.7 lineage was present in the US 5-6 weeks before its detection in the UK, yet as of January it comprised just 0.3% of cases nationally. After 2 months of circulation in California, the lineage is estimated to account for 0.4% of cases compared with 1.2% at a similar point in the UK. In Florida the lineage is associated with higher spread, 0.7% of cases, but this is not the situation in other US states.

These data emphasize that we cannot conclude that the B.1.1.7 lineage is biologically more transmissible. Multiple factors are likely at play, and this is why it is better to view the B.1.1.7 lineage variants and others in terms of their fitness – the reproductive success of the virus. Many factors can influence fitness, not just transmission. These could include increased physical stability of the particle, increased resistance to immune responses, longer duration of virus presence in the nasopharynx, increased infectious virus produced within the host, more efficient establishment of infection in a host, and more. A slight increase in any of these might drive a particular variant within a population but not actually affect person to person transmission. Whether such mutations are spread by founder effect – being in the right place at the right time – also must be taken into consideration.

The statistical models that have been used to approximate the transmission of SARS-CoV-2 variants cannot prove a biological property because drive through a population can be a consequence of various fitness parameters. Experiments either in animal models (in which case the relevance to humans is unknown) or measurement of infectious virus in humans is needed. So far none of the latter have been done for the current variants.

Immune control

A more immediate concern is whether any of the changes in spike protein within VOC impact the ability of immune response to control infections. This question has been directly addressed for neutralizing antibodies, e.g. those which can block infection. Antibodies recognize specific protein sequences on the virus particle, and specifically the spike protein for those given the mRNA vaccine. Some of the spike changes identified in variants are in regions known to bind antibodies. Consequently an important question is whether vaccination can inhibit infection with the variant viruses.

This question has been addressed for both the Moderna and the Pfizer mRNA vaccines. Sera from persons immunized with mRNA-1273 efficiently neutralized pseudotyped viruses bearing the SARS-CoV-2 spike glycoprotein from the B.1.1.7 lineage. These sera had a reduced (6.4 fold) neutralization titer when the South African B.1.351 lineage was used. However these sera still fully neutralized B.1.351 with a titer of 1:290 which may be sufficient to prevent severe COVID-19. Nevertheless, Moderna has announced that it will advance a modified vaccine (mRNA-1273.351) encoding the B.1.351 amino acid changes.

In a separate study, sera from individuals vaccinated with the Pfizer BNT162b2 mRNA vaccine was tested in neutralization assays using SARS-CoV-2 viruses with selected spike amino acid changes from the B.1.1.7 (deletion of amino acids 69/70, N501Y, D614G) or B.1.351 (E484K + N501Y + D614G) lineages. These changes had small effects on neutralization with the sera. However, the engineered viruses do not contain the full set of changes found in the B.1.1.7 and B.1.351 viruses, which might explain the different results compared sera with antibodies induced by mRNA-1273.

These observations provide confidence that the two mRNA vaccines will provide protection against COVID-19 caused by currently circulating variants. However genomic surveillance must be increased to ensure that any new spike changes that might arise are detected quickly and their effects on neutralization determined.

Disease severity

A previous study did not show evidence that viruses of the B.1.1.7 lineage were associated with an increased risk of hospitalization or death. However upon examination of additional data from three separate studies NERVTAG concludes that there is a ‘realistic possibility that infection with VOC B.1.1.7 is associated with an increased risk of death compared to infection with non-VOC viruses’. This conclusion was reached by statistical analyses of reported death rates among individuals infected with VOC B.1.1.7 or non-VOC viruses. For example, in one study the relative hazard of death was 1.35 (with a 95% confidence interval of 1.08-1.68). In another study the mean ratio of case fatality ratios between cases caused by VOC or non-VOC viruses was 1.36 (95% CI 1.18-1.56). These are small differences with large confidence intervals ranging from no effect to more effect, and the authors note that the absolute risk of death remains low. The statistics are computed by analyzing a limited dataset of all COVID-19 related deaths (8%) and consequently might be in error. Furthermore, there does not appear to be an increased risk of hospitalization associated with infection by VOC viruses. My reading of this report is that it mainly serves as a warning to continue genomic surveillance of variants with respect to death risk and does not come to a conclusion on causality.

Update: Novavax just released the first results of their phase 3, spike-protein based COVID-19 vaccine. Efficacy was nearly 90% in the UK, but in a smaller trial in South Africa it was 50% against the B.1.135 variant.

Biden’s pandemic plans

21 January 2021 by Vincent Racaniello

The Biden-Harris administration has released a document describing its plans to bring the United States out of the ‘worst public health crisis in a century’. It is a roadmap for not only ending the pandemic in the US, but to re-establish leadership in the global health care community and provide assistance to other countries. I highly recommend that everyone read it (link to pdf).

The National Strategy is organized around six goals, and they are all very ambitious in their scope. They will require a massive infusion of expertise and an expansion of the public health and scientific workforce. For example, the President will establish a US Public Health Jobs Corps to increase the public health workforce and bolster clinical care capacity for COVID-19. It is a stunning collection of multi-pronged plans which rival efforts to bring the US out of the Great Depression. As President Biden has said, the federal government alone cannot execute this plan: it will require the help of many Americans.

One part of the strategy that I would like to focus on is part of goal 3, which is to “mitigate spread through expanding masking , testing , treatment , data, workforce, and clear public health standards” and in particular, the section entitled “Prioritize therapeutics and establish a comprehensive, integrated COVID-19 treatment discovery and development program”. An excerpt is below:

This includes promoting the immediate and rapid development of therapeutics that respond to COVID-19 by developing new antivirals directed against the coronavirus family, accelerating research and support for clinical trials for therapeutics in response to COVID-19 with a focus on those that can be readily scaled and administered, and developing broad- spectrum antivirals to prevent future viral pandemics.

As I have written before, the COVID-19 pandemic could have been prevented by using broadly-acting anti-coronavirus antiviral drugs. I am very happy that the Biden Administration has received this information and plans to act on it. How this goal will be achieved remains to be seen. Presumably much of this research will be done through NIH funding, but that will require an increase in the budget of that agency. Will such an increase – at least 10 billion dollars a year – be approved by Congress? Such drug discovery will also require collaboration with industry. But industry does not develop drugs for which there is currently no market. How this problem will be overcome remains to be determined.

Successful execution of this strategy will require a strong focus on science and public health, and an expansion of the workforce in these areas. I have the feeling, after reading the Strategic Plan, that the Biden-Harris administration realizes that science is the way out of this pandemic, and that science will help us with future pandemics. This attitude is welcome after four years of anti-science anti-think. How well it will be realized is unsure, given the divided nature of both Congress and this Nation. As a scientist, it is some of the best news that I have seen in years.

Camelids for COVID

14 January 2021 by Vincent Racaniello

Human monoclonal antibodies that block infection with SARS-CoV-2 are being used to treat COVID-19 patients, but an alternative, antibodies produced in camelids (alpacas and llamas) might have advantages. Camelid monoclonal antibodies can be more cheaply produced in mass quantities in bacteria, and protein engineering can be quickly used to produce a better therapeutic product.

Human antibodies are large proteins made up of two heavy chains and two light chains (pictured). In contrast, camelid antibodies consist only of two heavy chains. Furthermore, the antigen binding domain (VHH in the figure) can be produced on its own in what is called a nanobody. Two VHH domains can be linked together to target separate epitopes (scFv in the figure).  VHH single domain antibodies lack the Fc domain, and therefore cannot bind Fc receptors, avoiding antibody-dependent enhancement.

To produce nanobodies against SARS-CoV-2, an alpaca and a llama were immunized with purified spike protein. Three nanobodies from the alpaca and one from the llama were identified that neutralize virus infection of cells in culture. The results of binding and structural studies revealed that three of the nanobodies recognize an epitope on the spike protein that is distinct from the site recognized by the other nanobody.

Three of the four nanobodies appear to block virus infection of cells by causing the spike protein to change to the post-fusion conformation, which is irreversible. The spike post-fusion conformation is usually attained upon binding to the cell receptor, ACE2, but the nanobodies can trigger fusion in the absence of this protein.

Multivalent nanobodies were engineered by joining VHH domains of two different specificities. These scFv proteins displayed 100-fold improved neutralization of SARS-CoV-2. Furthermore, while variant viruses resistant to neutralization by individual nanobodies were readily selected in cell culture, none were observed after virus passage in the presence of multivalent nanobodies. Delaying the emergence of nanobody resistant variants might lead to better therapeutic efficacy in patients.

Another attractive property of nanobodies is that they can be delivered to the respiratory mucosa by inhalation of aerosols. This delivery method could reduce the dose needed and allow treatment outside of medical facilities. In contrast, human monoclonal antibodies must be administered intravenously. 

Whether or not nanobodies help end the COVID-19 pandemic is unclear. However their clinical development should continue as they may be useful in the case of outbreaks in immunocompromised individuals who cannot be vaccinated. What is learned from their development against SARS-CoV-2 will likely save lives when the next pandemic inevitably arrives.

Musings of an anonymous, pissed off virologist

5 January 2021 by Vincent Racaniello

coronavirus

by Paul Bieniasz

Dr. Bieniasz is Professor and Investigator of the Howard Hughes Medical Institute at Rockefeller University.

As viruses go, SARS-CoV-2, is quite easy to neutralize with antibodies and, it turns out, straightforward to generate effective vaccines based on the spike protein. Perhaps, even probably, those two properties are causally related. Moreover, it appears that it is quite hard (albeit not impossible) to generate resistant spike variants that evade the polyclonal antibody responses elicited by said vaccines. This is all excellent news.

However, if I had a nefarious nature and wanted to ensure that the new SARS-CoV-2 vaccines were rendered impotent, these are a few things I would try.

First, we’d want to maximize the viral population size and diversity. Because SARS-CoV-2 has a proofreading polymerase, we might have to work hard to do this. The four measures outlined below might help accomplish this, assisting the virus to explore as much genetic diversity as possible, generating every conceivable point mutation as frequently as possible.

  • Delay the rollout of testing, so that the virus could spread undetected, seeding outbreaks in geographically, demographically and culturally diverse host populations, rendering it virtually impossible to quash with test-trace-isolate approaches.
  • Implement partial and patchy restrictions on movement and social interactions, thus maintaining consistently large pools of infected individuals.
  • Keep schools open, claiming that children don’t frequently transmit SARS-CoV-2. Because children have generally mild and perhaps more frequently asymptomatic infections, diversifying viral populations are more likely to spread undetected.
  • Start a rumor-mill, making full use of social media and other outlets, with topics such as masks are unnecessary or don’t work, that PCR tests are too sensitive or unreliable, that infection-induced ‘herd immunity’ is a reasonable strategy, or even that SARS-CoV-2 isn’t real. Undermining already inadequate public health measures helps keep viral population sizes large.

Second, during or after the establishment of large and diverse viral populations, we’d begin to apply selection pressure to enrich antibody resistance mutations. For that, we would elicit the help of the medical establishment to implement measures 5 and 6. They, laudably, want to help as many people as possible as quickly as possible — we could exploit this.

  • Treat tens of thousands of people with uncharacterized convalescent plasma of weak/unknown potency, without proper clinical trials, to get the ball rolling in applying some selection pressure to enrich for antibody resistant variants. (Again, I don’t know how effective this would be since it is mostly done in hospitals, where onward transmission would presumably be rare, but it would certainly be worth a try) Immunocompromised individuals with persistent infection might be especially helpful here.
  • Finally, and here’s the kicker: having developed a remarkable two-dose vaccine, that is extraordinarily effective, ADMINISTER IT TO MILLIONS OF PEOPLE – BUT DELAY THE SECOND DOSE. Generating a pool of hosts with just the right amount of neutralizing antibody to apply selection pressure, but also maintain sufficient levels of partially antibody-resistant virus to allow onward transmission is key here. We might not achieve this shortly after the first dose, but if we let immunity wane for a little while, say 4 to 12 weeks, we just might hit the sweet spot.

Of course, I don’t know if the above would be successful, but that’s what I’d try if I wanted to generate vaccine-resistant SARS-CoV-2 variants.

SARS-CoV-2 UK variant: Does it matter?

24 December 2020 by Vincent Racaniello

A variant of SARS-CoV-2 has been spreading within England, and it has been suggested that this virus is more transmissible. In this video Vincent explains the properties of the SARS-CoV-2 UK variant and why claims that it is more transmissible are not supported by experimental data.

This video was recorded on Monday, 21 December 2020. Since then additional data on the UK variant have been released but they do not change my view: there are no biological data in humans showing that the virus has increased transmissibility. The variant is certainly spreading in the population, but that could be due to other situations that have not been ruled out.

Encouraging clinical data for universal flu vaccine candidate

17 December 2020 by Vincent Racaniello

by Helen Stillwell

The results from a phase I clinical trial to test the safety and immunogenicity of a universal flu vaccine candidate reported encouraging results – strong titers of broad and functional antibodies persisted for over a year in healthy adults following vaccination. 

Influenza viruses contain segmented RNA genomes. The viral envelope contains two types glycoproteins or ‘spikes’ that facilitate viral entry into host cells – hemagglutinin (HA) and neuraminidase (NA). Typically, HA and NA proteins stud the viral envelope at a ratio of four to one. Additionally, there are three types of influenza virus – A, B, and C. Influenza A viruses are further characterized by subtype based on their HA and NA proteins. Three HA subtypes (H1, H2, H3) and two NA subtypes (N1, N2) have be shown to cause widespread influenza transmission in humans.

The HA molecule contains two structural regions: the head and stalk. Spatially, the head is more prominent than the stalk, and antibodies to the head of the HA molecule have been shown to neutralize viral infectivity. The structural and functional characteristics of influenza viruses allow for antigenic drift and antigenic shift. Antigenic drift results when influenza virus strains develop frequent amino acid changes in the head domain, and the strain eventually evolves into one that can no longer be neutralized by antibodies to the parent virus. Antigenic shift occurs when an influenza A virus acquires the HA domain (and potentially the NA domain) from a different viral subtype. 

The combination of these phenomena requires that flu vaccines be reassessed each year based on the viral strains currently circulating in the human population. These vaccines are efficacious when they are well matched to the circulating strains; however, mismatches are not uncommon. As such, vaccinologists have been seeking to develop a ‘universal’ flu vaccine that would confer protection against all seasonal, zoonotic, and emerging pandemic influenza viruses. 

Current seasonal influenza vaccines primarily target the HA head domain of the circulating strains. However, as mentioned previously, the head domain can escape neutralization by accumulating sufficient mutations through antigenic drift. The HA stalk domain, however, is more conserved; therefore, researchers hypothesized that a vaccine targeting the HA stalk domain might offer protection independent of antigenic drift or shift. 

To provoke an immune response to the HA stalk domain, researchers at Mount Sinai developed a sequential vaccine strategy by generating chimeric HA (cHA) proteins consisting of conserved stalk and head domains from various avian influenza subtypes. Most adults already possess immune memory to the H1 HA domain as well as antibodies and memory B cells specific to the stalk domain; therefore, it was proposed that vaccinating individuals with cHA constructs that consist of head domains from different avian influenza virus subtypes and a conserved stalk domain might redirect the immune response to the stalk.

The clinical trial consisted of 5 different treatment groups:

Group 1: LAIV8-IIV5/AS03

Day 1: Intranasal (i.n.) live-attenuated influenza virus (LAIV) vaccine expressing ch8/1 HA and an N1 NA.
Day 85: Intramuscular (i.m.) inactivated influenza virus (IIV) vaccine expressing ch5/1 HA and an N1 NA with an adjuvant (AS03). 

Group 2: LAIV8-IIV5

Day 1: (i.n.) LAIV vaccine expressing ch8/1 HA and an N1 NA.
Day 85: (i.m.) IIV vaccine expressing ch5/1 HA and an N1 NA with no adjuvant. 

Group 3: Placebo Control 1

Day 1: (i.n.) Saline solution.
Day 85: (i.m.) PBS 

Group 4: IIV8/AS03-IIV5/AS03

Day 1: (i.m.) IIV vaccine expressing cH8/1N1 with AS03.
Day 85: (i.m.) IIV vaccine expressing cH5/1N1 with AS03. 

Group 5: Placebo Control 2

Day 1: (i.m.) PBS
Day 85: (i.m.) PBS

The regimen for Groups 1 and 3 comprised live-attenuated influenza virus (LAIV) followed by an inactivated influenza virus (IIV). This combination was tested based on previous studies showing that it had provoked optimal antibody responses with influenza virus vaccines in humans and non-human primates and with chimeric HA-based vaccines in ferrets. This combination had also been found to convey better protection against infection than the chimeric HA-based IIV-IIV regimen in ferrets; therefore, it was hypothesized that the intranasal LAIV followed by the intramuscular IIV boost might confer better protection than the two intramuscular doses of IIV (Group 4).

Ultimately, the study reported that Group 1 participants did not produce significant anti-stalk antibody titers after the day 1 LAIV dose; however, when these participants were boosted with IIV5/AS03 they induced a strong anti-stalk antibody response. When the boost was given without an adjuvant in Group 2, lower anti-stalk antibody titers were observed. In Group 4, however, the initial administration of IIV8/AS03 induced a very strong anti-stalk antibody response. Although these titers dropped slightly between days 25 and 85, they increased again after the administration of IIV5-AS03 on day 85. Further, these antibody titers persisted above baseline levels at 420 days after vaccine administration. While Groups 1 and 2 antibodies also persisted, they did so at lower levels. Anti-stalk antibodies did not increase in the placebo groups over the course of the study, and no significant adverse reactions to the vaccine were reported. Additionally, mice treated with post vaccination serum were protected from viral challenge as compared to mice treated with pre-vaccination serum. Although one might have predicted Groups 1 and 2 to show better protection than Group 4 based on the data established in ferrets, animal models do not always approximate vaccine responses in humans.Though encouraging, these results are still preliminary, and future trials will shed light on whether antibodies to the stalk protein will prove to be as protective as those elicited by natural infection. It may take several years to develop the diversity of chimeric hemagglutinins needed to make a universal flu vaccine, and later phase clinical trials to test the vaccine’s efficacy and superiority to existing vaccines would need to be conducted. This study does demonstrate, however, that “you can develop a vaccine strategy that produces stalk-reactive antibodies in humans,” said virologist Florian Krammer in an interview for Science, one of the lead investigators on the project. It represents an important step towards developing a universal vaccine strategy that could replace the seasonal model.

Image credit: Principles of Virology, ASM Press

TWiV 693: Vax to the future

16 December 2020 by Vincent Racaniello

On this episode, FDA EUA for Pfizer mRNA vaccine, efficacy of AstraZeneca ChAdOx1 COVID-19 vaccine, and an orally administered drug that blocks SARS-CoV-2 transmission in ferrets.

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Brianne Barker

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Download TWiV 693 (69 MB .mp3, 115 min)
Subscribe (free): iTunes, Google Podcasts, RSS, email

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Show notes at microbe.tv/twiv

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