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Antibodies Against SARS-CoV-2 Nucleocapsid Protein May Not Be Reliable Markers for Infection in Vaccinated People

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by Gertrud U. Rey

You are fully vaccinated against SARS-CoV-2 and have presumably never been infected with the virus. But how can you know for sure? One way to find out is by testing your blood for the presence of antibodies against the viral nucleocapsid protein, which can only be encountered during natural infection. This is because all of the SARS-CoV-2 vaccines used in the U.S. only encode the viral spike protein (none encode nucleocapsid [N] protein), and thus they only stimulate production of antibodies against spike. This approach differentiates between vaccine- and virus infection-induced antibodies and allows one to accurately determine whether a vaccinated person was naturally infected. Or so we thought until now.

Two recent letters to the editor of the Journal of Infection note that not every natural infection induces production of anti-nucleocapsid (or, “anti-N”) antibodies. The letters cast doubt on whether these antibodies are reliable markers for a prior SARS-CoV-2 infection.

The authors of the first letter measured antibody responses in 4,111 vaccinated and 974 unvaccinated Irish healthcare workers. Only 23 of the vaccinated participants, all of whom had received two doses of the Pfizer mRNA vaccine, experienced a SARS-CoV-2 infection at some time after vaccination. As expected, each of the 23 individuals had antibodies against the spike protein, but surprisingly, only six (26%) had detectable anti-N antibodies. In contrast, 82% of unvaccinated participants with a previous PCR-confirmed infection had detectable anti-N antibodies. This result suggests that anti-N antibodies may not be the most accurate indicators of a prior natural infection in vaccinated people; and it further implies that vaccinated individuals may neutralize incoming viruses early during infection, thus preventing and/or limiting their ability to develop antibodies against nucleocapsid protein.

The second letter, which was written in response to the first letter, confirmed and further substantiated these results. Citing data from serosurveys done in Japan, the authors showed that patients who were infected within two months of a third dose of the Pfizer mRNA vaccine were less likely to experience COVID-19 symptoms than patients who were infected 4-8 months after the third dose. These findings are in line with our current understanding of sterilizing immunity, a type of immunity that prevents both disease and infection, which appears to occur most often during the months following vaccination, when high levels of vaccine-induced antibodies probably sequester an incoming virus before it has a chance to infect cells. The authors also showed that participants infected within two months of their third vaccine dose had significantly lower levels of anti-N antibodies than those infected several months later. Although this result seems surprising at first, it actually further supports the notion that vaccination only induces sterilizing immunity for a short time after vaccination, when existing vaccine-induced anti-spike antibodies neutralize incoming virus before the immune system has a chance to respond to the virus and produce antibodies specific to the nucleocapsid protein.

The authors of both letters further mention that COVID-19 patients who experienced symptoms were more likely to have detectable anti-N antibodies than were patients without symptoms, an observation that is in agreement with serological surveys done before vaccines became available. This finding suggests that patients who developed symptoms did not have sterilizing immunity and were subject to a productive viral infection that led to the development of symptoms and production of antibodies to nucleocapsid and other viral proteins.

These two studies provide an interesting perspective of antibody responses to SARS-CoV-2 infection in vaccinated people, and they may inform better strategies for gauging infection after vaccination.

One and Done

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by Gertrud U. Rey

On February 27, 2021, the FDA issued an emergency use authorization for a third SARS-CoV-2 vaccine. The vaccine was developed by Janssen Pharmaceutica, a Belgium-based division of Johnson & Johnson, in collaboration with Beth Israel Deaconess Medical Center in Boston. Perhaps the most exciting feature of this new vaccine is that it only requires one dose to be effective in inducing an immune response.

The vaccine is named Ad26.COV2.S because it consists of a human adenovirus vector, with a DNA genome, into which has been inserted the gene that encodes the full-length SARS-CoV-2 spike protein (pictured). Ad26.COV2.S is similar to AstraZeneca’s vaccine, based on a different adenovirus, and with a slightly different version of spike, which is not yet authorized in the U.S. The notion of using a virus as a vector to deliver vaccines to humans is based on the ability of viruses to enter cells by attaching to host cell receptors and releasing their genome into the cell. Upon injection into a vaccine recipient, the vaccine vector should enter cells and serve as a code for host proteins to synthesize the SARS-CoV-2 spike protein from the inserted gene. Ideally, the spike protein will then act as an antigen to prime the immune system to recognize SARS-CoV-2 if it infects the body at a later time. 

Adenoviruses are particularly suitable as vectors for delivering foreign genes into cells because they have a double-stranded DNA genome that can accommodate relatively large segments of foreign DNA, and because they infect most cell types without integrating into the host genome. However, because of the prevalence of adenovirus infections in humans, most people have adenovirus-specific antibodies that could bind and neutralize these vectors, thus rendering them less effective at stimulating antibodies to the inserted gene product. AstraZeneca circumvented this issue by using an adenovirus of chimpanzee origin that does not normally infect humans. The adenovirus used to make Ad26.COV2.S (Adenovirus 26) is of human origin; however, when tested, most people have very few antibodies that inactivate this adenovirus, compared to antibodies against other adenoviruses. Thus, potential Ad26.COV2.S recipients are less likely to have pre-existing antibodies to the adenovirus vector itself. To optimize Adenovirus 26 for use as a vaccine vector, Janssen investigators deleted the gene that regulates viral replication, thus ensuring that the virus vector cannot cause an infection in human cells.

During infection, the SARS-CoV-2 viral particle fuses with the host cell membrane; a process that is mediated by two main events: 1) a structural rearrangement of the spike protein from its pre-fusion conformation; and, 2) cleavage of the spike protein by a cellular enzyme called furin. Based on the knowledge that the pre-fusion, uncleaved form of spike is more stable and immunogenic, Janssen investigators also inserted two mutations into the spike gene: one that locks the translated spike protein into its pre-fusion conformation, and one that prevents its cleavage by furin.  

The FDA’s decision to issue an emergency use authorization for Ad26.COV2.S was based on safety and efficacy data from an ongoing Phase III clinical trial done in 39,321 participants who received either a single dose of Ad26.COV2.S or a placebo control. The trial was randomized, meaning that participants were randomly assigned to the experimental group receiving the Ad26.COV2.S vaccine, or the control group, so that the only expected differences between the experimental and control groups were the outcome variables studied (safety and efficacy). Randomizing trial participants eliminates unwanted effects that have nothing to do with the variables being analyzed. The trial was also double-blinded, meaning that neither the investigators nor the subjects knew who was receiving a particular treatment. Double-blinding leads to more authentic conclusions because they reduce researcher bias.

The basic findings of the trial were as follows:

  • side effects related to vaccination were mild to moderate; and
  • the vaccine was
    • 66% effective at preventing moderate to severe COVID-19 across all geographic areas and age groups (U.S., South Africa, and six countries in Latin America);
    • 72% effective at preventing moderate to severe COVID-19 across all age groups in the U.S.; 
    • 85% effective at preventing severe disease; and
    • 100% effective at preventing COVID-19-related hospitalization and death as of day 28 after vaccination.

The apparently reduced efficacy of Ad26.COV2.S compared to the Moderna and Pfizer vaccines has led to considerable public skepticism. However, this is an unfair comparison for several reasons. Ad26.COV2.S was tested at a time when more variants were in circulation, including in places where the Moderna/Pfizer vaccines are thought to be less effective against locally circulating variants. Some limited data also suggest that Ad26.COV2.S might protect from asymptomatic infection and may thus prevent transmission from vaccinated individuals to non-vaccinated individuals. Although there is some evidence to suggest that the Pfizer vaccine has a similar effect, no such data exist yet for the Moderna vaccine.

The most critical measure of a vaccine’s efficacy is how well it prevents severe disease, hospitalizations, and deaths, and in this regard, all three vaccines are comparable. Moreover, Ad26.COV2.S has at least two advantages over the Pfizer/Moderna vaccines: 1) it does not require a freezer and can be stored in a refrigerator for up to three months; and, 2) it can be administered in a single dose. This will increase vaccine uptake, because people won’t have to get two shots and/or remember to get the second shot.  It also makes it easier to immunize people with limited access to healthcare, such as the homeless and people living in remote areas. When all these factors are considered together, it is clear that Ad26.COV2.S will be a crucial additional tool in the fight against this pandemic.

TWiV 690: This is your brain on SARS-CoV-2

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TWiV reviews the difficulties in predicting species susceptibility to SARS-CoV-2 infection by only examining the ACE2 protein, and the olfactory mucosa as a portal of entry into the central nervous system in COVID-19 patients.

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Show notes at microbe.tv/twiv

Antiviral antibodies in COVID-19 patients

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coronavirus Spike

In infectious disease world, the term “correlate of protection” refers to something that can be measured – antibodies or T cells – which indicates that a person is protected against infection or disease. The correlates of protection for SARS-CoV-2 infection of humans are not yet known – after all it is still early in this pandemic – but many laboratories are investigating whether anti-viral antibodies are the key.

A major assumption of these studies, and indeed of most of the vaccines in development, is that antibodies to the SARS-CoV-2 spike glycoprotein are important for protection. The logic for this is based on several lines of evidence, including the fact that spike binds the cell receptor, ACE2, and so blocking spike prevents infection; and immunization of animals with spike-encoding vaccines, or administration of anti-spike antibodies can prevent infection with SARS-CoV-2 . Vaccine-induced antibodies are clearly a correlate of protection in nonhuman primates. Whether antibodies to spike are protective in humans remains to be determined, but we should have the answer within the year, from the results of vaccine efficacy trials and observing whether natural infection broadly confers protection against reinfection.

Another way to asses the roles of antibodies in protection against COVID-19 is by studying their levels and their durability in patients. Three recent papers have addressed these questions in different ways.

A study of sera from 30,082 individuals with mild to moderate COVID-19 reveals that all have moderate to high titers of antibodies that bind the viral spike protein. The ability of these antibodies to neutralize SARS-CoV-2 infectivity was studied in only 120 samples. While neutralization titers correlated with spike-binding titers, it is not clear if this pattern applies to the entire patient set. Authors report that neutralization titers are stable for at least 5 months, and argue that this longevity is a consequence of long-lived memory B cells in the bone marrow. Whether this assertion is correct must be determined experimentally.

A 3-month study of 65 patients with COVID-19 ranging from asymptomatic to severe also reveals anti-SARS-CoV-2 antibody responses in most patients when sampled beyond 8 days after symptom onset. Neutralizing antibody titers decline within 3 months, often to baseline levels in patients who had less severe disease.

A third study followed 97 non-hospitalized patients for 8 weeks. All produced neutralizing antibodies at different levels which decreased by 45% over 4 weeks.

The results of these studies differ, with the first claiming that anti-SARS-CoV-2 neutralizing antibodies are durable as a consequence of memory B cells, and the other two finding a more rapid decline. Which is correct is not clear as the patient samples and methodologies are different. The question of whether mild to moderate COVID-19 is associated with humoral memory remains an open question. We do know from a recent study that B cell memory is not present in patients with severe disease, as a consequence of lack of germinal centers in lymph nodes and spleen. If B cell memory is established after mild to moderate infection, then reinfection should lead to rapid anti-viral antibody production and possible protection against disease.

Similar studies over longer periods of time are needed to determine if anti-viral antibody levels eventually decline to baseline, implying lack of B cell memory, or if they remain at low levels as they are replenished by long-lived plasma cells in the bone marrow.

No evidence for increased human transmission of SARS-CoV-2

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coronavirus Spike

Don’t believe the headlines that SARS-CoV-2 is becoming more transmissible! Virologists are making conclusions that are not justified by the data.

SARS-CoV-2 virus isolates from many parts of the world have a single amino acid change in the spike protein, D614G. This observation in itself doesn’t mean very much. It could be that the change arose early in the outbreak and as this virus spread to other areas it was maintained because it has no fitness cost. We call this behavior ‘founder effect’. Whether the change has been selected for – what we would call ‘positive selection’, as claimed by many virologists, needs to be proven by experiments that have not been done.

The D614G change appears to make the virus more infectious in cells in culture. However, there are limitations to the conclusions that may be drawn from these data. First, cells that have been used are irrelevant to human transmission, such as a kidney cell line (VERO) from Vervet monkeys. I would like to see data from cultures of human respiratory epithelial cells, including airway-liquid cultures and lung organoids. Second, SARS-CoV-2 was not used for these experiments, but pseudotyped viruses – recombinant retroviruses or vesicular stomatitis virus with the SARS-CoV-2 spike glycoprotein gene inserted. The reason for this approach is that working with SARS-CoV-2 requires a BSL-3 laboratory which is not easy to come by. Nevertheless, even if all these issues were addressed, do we really think that results in cell culture tell us anything about human to human transmission? Seriously, virology colleagues? A monolayer of cells in culture in no way compares with the architecturally complex respiratory epithelium with mucus, antibodies, innate and adaptive responses.

I don’t mention using an animal model to assess SARS-CoV-2 transmission potential because there are no good models (yet) for animal to animal transmission of the virus.

I can think of experiments that could be done to determine if viruses with D614G behave differently in humans, but they have not been done. One approach would be to determine if the change leads to higher shedding of infectious virus from the upper tract. One study reported higher levels of viral RNA in the upper tract of patients infected with 614G compared with patients infected with 614D. This observation is meaningless, because everyone knows that nucleic acids detected by PCR does not always mean that infectious virus is present. Increased RNA might be a consequence of a change in the viral RNA polymerase caused by another mutation that accompanies the spike change. What needs to be done is to measure the levels of INFECTIOUS VIRUS shed from the upper respiratory tract of humans infected with either variant. Even then I would argue that the results cannot be interpreted, because we do not know how much of an increase in virus shedding would lead to an increase in transmission. Would twofold more virus be enough? Fivefold? Tenfold? A thousand fold? Anyone who says they know is wrong.

We know that the dispersion factor, k, of SARS-CoV-2 is 0.1, which means that about 10% of cases lead to 80% of spread. Which virus are these individuals transmitting, 614D or 614G? The results would contribute to an understanding of the role of this amino acid change in transmission.

It’s unfortunate that during a serious outbreak, rigorous science appears to be relegated to the back seat. You might remember during the 2015 Ebolavirus outbreak in West Africa, a viruses with a single amino acid change in the spike protein arose early and predominated. This change made the virus more infectious in cells in culture in the laboratory, but was never shown to have any effect on human transmission. In a nonhuman primate model, the change actually reduced virulence of the virus. Whether the predominance of virus isolates with the amino acid change was a founder effect or positive selection was never determined.

An important consideration is to identify the selection pressure for SARS-CoV-2 viruses with increased transmission. By the time the virus was detected in China late in 2019, it was already very good at transmitting among humans. I fail to see what selection pressure would lead to the emergence of such a variant.

The D614 change does not appear to change the virulence of SARS-CoV-2, nor its ability to be neutralized by antibodies. Let’s toss all the above arguments aside and assume that D614G increases human transmission. What should we do? Wear face masks, avoid large gatherings of people, embrace physical distancing. All of which are already being done – or are they?

A mouse model for COVID-19

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coronavirus Spike

Small animal models (mice, ferrets, hamsters) are essential for understanding how SARS-CoV-2 causes disease and for pre-clinical evaluation of vaccines and antiviral therapeutics. One promising model has been developed by modifying SARS-CoV-2 so that it binds to murine ACE2.

Laboratory mice cannot be infected with SARS-CoV-2 because the viral spike glycoprotein (illustrated) does not bind to murine ACE2, the ortholog of the human receptor. Mice transgenic for the human ACE2 gene, produced after the SARS-CoV outbreak, can be infected with SARS-CoV-2, but the pathogenesis does not resemble that observed in humans. For example, mortality in infected mice appears to be a consequence of replication in the central nervous system, not in the respiratory tract. The altered pathogenesis might be due to improper expression patterns of the ACE2 gene.

In another approach to producing a mouse model for COVID-19, the spike glycoprotein of SARS-CoV-2 was modified so that it efficiently binds murine ACE2 protein. Only two amino acid changes in the S protein were required to allow infection of cells that produce murine ACE2 without affecting reproduction in monkey cells.

Upon intranasal infection of young mice, virus replication was detected in the upper and lower airways and was accompanied by mild to moderate disease. In contrast, after inoculation of older mice, viral replication was not only detected in the upper and lower tract but was associated with lung inflammation and loss of pulmonary function. The age-dependency of disease mimics what is observed in humans.

As a proof of principle, the mouse model was used to evaluate efficacy of a vaccine and antiviral. After vaccination of mice with a Venezuelan equine encephalitis vector carrying the SARS-CoV-2 spike protein gene, reduced virus replication in lung and upper respiratory tract was observed after challenge compared with control mice. Furthermore, prophylactic and therapeutic administration of IFN-lambda-1 reduced virus titers in the lung compared with untreated mice.

This mouse model is attractive because it does not require the development of genetically altered animals. However it is not a perfect model because there are likely differences in tissue distribution of ACE2 in mice and humans which could affect disease outcomes. Whether this difference has an impact on the usefulness of the mouse model remains to be seen. Meanwhile other mouse models are being developed which might have other advantages: for example the production of human ACE2 in mice by using adenovirus-associated virus vectors.