sterilizing immunity

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

3 November 2022 by Gertrud U. Rey

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.

Herd Immunity and this Pandemic

2 June 2022 by Gertrud U. Rey

by Gertrud U. Rey

Photo courtesy of Andrea Lightfoot photography

Herd immunity occurs when a large enough percentage of the population has acquired either natural or vaccine-induced immunity against an infectious disease, thereby indirectly protecting a minority of non-immune individuals who are dispersed throughout the population. During this pandemic, many prominent scientists have stated that it is impossible to achieve herd immunity in the context of COVID-19, leading some to conclude that a mass SARS-CoV-2 vaccination campaign would be pointless. However, this thinking is flawed, and I want to explain why.

Traditionally, herd immunity is thought to create a barrier for the transmission of infectious agents, resulting not only in prevention of disease, but also prevention of infection. This understanding was based on previous observations that vaccination against poliovirus, measles virus, and other pathogens led to drastic reductions in the incidence of disease burden. It is reasonable to assume that if there is no disease, there is probably also no virus; and hence no viral infection or transmission of virus. However, past vaccination campaigns were not followed up with regular testing programs, so we actually have no way of knowing whether vaccination prevented infection and transmission! Considering that the vast majority of poliovirus infections are asymptomatic, it is possible that some polio virus infections and transmission occurred even after vaccination, despite the fact that those infections did not lead to disease.

The widespread testing measures adopted during the present pandemic have revealed the approximate frequency of asymptomatic SARS-CoV-2 infections, giving us a clearer understanding of the difference and dynamics between disease and infection. The type of immunity that prevents both disease and infection is called sterilizing immunity, and it is mostly thought to be induced by neutralizing antibodies, which inactivate infectious agents before they have a chance to infect a cell, thereby directly neutralizing the biological effect of the agent. However, any immune activity that prevents replication of a pathogen directly or indirectly necessarily induces sterilizing immunity, including the activity of non-neutralizing antibodies, whose binding can trigger other immune functions that can also prevent infection and replication.

Do SARS-CoV-2 vaccines induce sterilizing immunity? The answer to this question is complicated. There are many studies showing that most people have high levels of antibodies in the months following vaccination, and this large proportion of circulating antibodies could likely sequester an incoming virus before it has a chance to enter cells, infect them, and replicate. In this sense, the SARS-CoV-2 vaccines do induce sterilizing immunity, but only within a certain time period after vaccination. As antibody levels contract over time (a normal process), they leave behind a baseline population of memory B cells that can quickly expand and mass-produce new antibodies upon a subsequent encounter with SARS-CoV-2. Likewise, memory T cells can quickly react to incoming virus and virus-triggered signals, and destroy infected cells. Therefore, it is likely that when circulating SARS-CoV-2-specific antibody levels decline months and years after vaccination, the collective activity of memory immune cells will protect one from disease, but probably not infection, meaning that the SARS-CoV-2 vaccines no longer induce sterilizing immunity at that time. In other words, vaccinated people could briefly replicate and transmit low levels of virus, at least until memory immune responses kick in, which then prevent illness and additional viral replication and spread.

The emergence of new variants that are not as well recognized by existing vaccine-induced antibodies may also allow for some increased viral transmission, thus slowing down the establishment of immunity in the population. However, immune responses are not binary, and even a low level immune response that doesn’t protect against infection and spread but prevents serious disease can play a critical role in slowing down the pandemic. Vaccination has historically been very effective at suppressing community outbreaks, despite the fact that most vaccines do not induce sterilizing immunity.

Vaccination or natural immunity do not have to prevent all infections, and immunity does not have to last a lifetime for a pandemic to end. Pediatrician and vaccinologist Paul Offit defines herd immunity as the point where the serious disease burden is reduced sufficiently so as to no longer overwhelm the healthcare system. It’s becoming pretty clear that the pandemic is slowing down in the US, especially in the context of severe disease, hospitalization, and death; and that this is likely due to increased SARS-CoV-2 immunity among US residents. It is therefore likely that reduced illness and a shortened period of transmission from immune individuals will also reduce the overall rate of community infection and transmission. And in the end, it doesn’t matter whether we call it herd immunity, community immunity or some other name; the pandemic will end because a majority of the population is no longer susceptible to severe COVID-19.

[Please check out my video Catch This Episode 29 for an explanation of sterilizing immunity.]

The Route Matters

3 September 2020 by Gertrud U. Rey

by Gertrud U. Rey

There are currently 315 therapeutic drugs and 210 vaccine candidates in development to treat or prevent SARS-CoV-2 infection. Many of these vaccines are designed to be administered by injection into the muscle.

Intramuscular injection of a vaccine antigen typically induces a systemic (serum) immune response that involves the action of IgM and IgG antibodies. IgM antibodies appear first and typically bind very strongly to antigens, to the extent that they often cross-react with other, non-specific antigens. IgG antibodies arise later, are a lot more specific than IgM, and provide the majority of antibody-based immunity against invading pathogens. Intramuscular immunization usually does not induce very high levels of serum IgA, a type of antibody that is more prevalent in mucosal surfaces and represents a first line of defense against invasion by inhaled and ingested pathogens. The role of IgA in the serum is mostly secondary to IgG, in that IgA mediates elimination of pathogens that have breached the mucosal surface.

Several of the SARS-CoV-2 vaccine candidates currently in clinical trials consist of a replication-deficient adenovirus with an inserted gene that encodes a SARS-CoV-2 antigen. The suitability of adenoviruses as vectors for delivering foreign genes into cells was discussed in a previous post, which summarized preliminary phase I/II clinical trials assessing the safety and efficacy of a chimpanzee adenovirus-vectored replication-deficient SARS-CoV-2 vaccine candidate encoding the full-length SARS-CoV-2 spike protein (AZD1222). The spike protein has been the primary antigenic choice for a number of SARS-CoV-2 vaccine candidates because it mediates binding of the virus to the ACE2 host cell receptor via its receptor-binding domain (RBD), and it also mediates fusion of the viral particle with the host cell membrane via its fusion domain. Both of these spike domains are highly immunogenic and are targeted by neutralizing antibodies, which bind viral antigens, inactivating virus and preventing infection of new cells. However, preliminary results suggest that AZD1222 only protects against SARS-CoV-2 lung infection and pneumonia but doesn’t appear to prevent upper respiratory tract infection and viral shedding.

To mediate fusion of the virus particle to the host cell membrane, the SARS-CoV-2 spike protein undergoes a structural rearrangement from its pre-fusion conformation. Because the pre-fusion form is more immunogenic, vaccines encoding the spike protein often contain a mutation that locks the translated spike protein into this pre-fusion structure. In a recent publication, virologist Michael Diamond and colleagues analyzed the efficacy of an adenovirus-vectored SARS-CoV-2 vaccine candidate and compared its protective effects after intramuscular injection to those after administration by the intranasal route. The vaccine, named ChAd-SARS-CoV-2-S, is similar to AZD1222 except that its spike gene encodes the pre-fusion stabilized spike protein. To assess the antibody responses induced by intramuscular vaccination with ChAd-SARS-CoV-2-S, the authors injected mice with 10 billion viral particles of either ChAd-SARS-CoV-2-S or a control vaccine consisting of the same adenovirus shell, but lacking the spike protein gene insert. They found that one dose of ChAd-SARS-CoV-2-S induced strong serum IgG responses against both the entire spike protein and the RBD, but no IgA responses in the serum or in mucosal lung cells.

While antibodies are an important part of the adaptive immune response, cell-mediated immunity is just as important and at the very least results in activation of white blood cells that destroy ingested microbes and also produces cytotoxic T cells that directly kill infected target cells. During a first exposure to a pathogen, T helper cells typically sense the presence of antigens on the surface of the invading pathogen and release a variety of signals that ultimately stimulate B cells to secrete antibodies to those antigens and also stimulate cytotoxic T cells to kill infected target cells. Analysis of these T cells in mice immunized with one or two doses of intramuscularly administered ChAd-SARS-CoV-2-S revealed that two vaccine doses induced both T helper and cytotoxic T cell responses against the whole spike protein. Collectively, these results suggest that although intramuscular vaccination produces strong systemic adaptive immune responses against SARS-CoV-2, it induces little, if any, mucosal immunity.

To determine whether intramuscular immunization with ChAd-SARS-CoV-2-S protects mice from infection, the authors intentionally infected (“challenged”) immunized mice with SARS-CoV-2. Although a single vaccine dose protected the mice from SARS-CoV-2 infection and lung inflammation, the mice still had high levels of viral RNA in the lung after infection, suggesting that intramuscular administration of the vaccine does not lead to complete protection from infection.

In an effort to see whether vaccination by the intranasal route provides more complete protection, the authors inoculated mice with a single dose of ChAd-SARS-CoV-2-S or control vaccine through the nose. Analysis of serum samples and mucosal lung cells four weeks after vaccination revealed that recipients of ChAd-SARS-CoV-2-S had high spike- and RBD-specific levels of neutralizing IgG and IgA in both the serum and the lung mucosa, and that the number of B cells producing IgA was about five-fold higher than that of B cells producing IgG. Interestingly, the neutralizing antibodies were also able to inactivate SARS-CoV-2 viruses containing a D614G change in the spike protein, suggesting that ChAd-SARS-CoV-2-S can effectively protect against other circulating SARS-CoV-2 viruses. Intranasal vaccination also induced SARS-CoV-2-specific cytotoxic T cells in the lung mucosa, specifically T cells that produce interferon gamma, an important activator of macrophages and inhibitor of viral replication.

The ideal immune response is “sterilizing” – meaning that it completely protects against a new infection and does not allow the virus to replicate at all. To evaluate the ability of a single intranasal dose of ChAd-SARS-CoV-2-S to induce sterilizing immunity, the authors analyzed immunized and infected mice for serum antibodies produced against the viral NP protein. Because the vaccine does not encode the NP protein, any antibodies produced against this protein would be induced by translation of the NP gene from the challenge virus and active replication of the virus. All of the mice immunized with a single dose of intranasally administered ChAd-SARS-CoV-2-S had very low levels of anti-NP antibodies compared to recipients of the control vaccine, suggesting that ChAd-SARS-CoV-2-S induced strong mucosal immunity that prevented SARS-CoV-2 infection in both the upper and lower respiratory tract. This means that if intranasally immunized mice were to be exposed to SARS-CoV-2, they would not be able to replicate the virus or transmit it to others.

The study has some notable limitations. First, it is well known that mice can be poor predictors of human disease outcomes. Second, because the mouse ACE2 receptor doesn’t easily bind SARS-CoV-2, the mice were engineered to express the human ACE2 receptor, which added a further artificial variable to an already imperfect model system. Third, it is presently unknown how long the observed immune responses would last. That being said, studies with influenza virus have shown that mucosal immunization through the nose can elicit strong local protective IgA-mediated immune responses. Further, there are clear advantages to intranasal vaccine administration: inoculation is simple, painless, and does not require trained professionals. The adequacy of a single dose would also lead to more widespread compliance. Lastly, a vaccine that prevents viral shedding would be ideal, because in addition to preventing disease in the exposed individual, it would prevent transmission to others.

None of the SARS-CoV-2 vaccine candidates currently in clinical trials are delivered by the intranasal route. If the results observed in these mouse experiments can be duplicated in humans, ChAd-SARS-CoV-2-S would clearly be superior to other SARS-CoV-2 vaccine candidates.