virology blog

About viruses and viral disease

IgG

An Intranasal SARS-CoV-2 Vaccine Candidate

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by Gertrud U. Rey

There are numerous advantages to administering a vaccine by the intranasal route: it is simple, painless, and does not require special training. The last time I discussed SARS-CoV-2 vaccine candidates for potential intranasal delivery, the idea was still fairly new. However, multiple new studies are now showing that delivery of a SARS-CoV-2 vaccine into the nose induces immune responses that are superior to those observed after delivery into the muscle.

The authors of one such recent study compared the efficacy of a single dose of two different adenovirus-based SARS-CoV-2 vaccine candidates after intramuscular injection to that after administration by the intranasal route. Both vaccines encoded three viral antigens: 1) the full-length S1 domain of the SARS-CoV-2 spike protein, which contains the region that binds to the host cell receptor ACE2; 2) the full-length nucleocapsid protein, which is essential for binding and packaging the viral genome; and 3) a truncated version of the RNA-dependent RNA polymerase (RdRp), the enzyme responsible for copying the viral genome during infection. The three antigens were inserted into either one of two adenovirus vectors: a virus of human origin to produce “Tri:HuAd” or a virus of chimpanzee origin to produce “Tri:ChAd.” Most currently approved SARS-CoV-2 vaccines only encode the full-length spike protein, but not nucleocapsid or RdRp. These two additional genes were likely included in the present study because nucleocapsid is the most abundant viral protein in infected cells, and because RdRp is well conserved among coronaviruses, thus potentially leading to broader immune responses.

To assess the antibody responses induced by either vaccine candidate, the authors immunized mice with a single dose of Tri:HuAd or Tri:ChAd into the nose or into the muscle and collected serum and lung wash samples 2, 4 and 8 weeks later. The collected samples were then tested for the presence of IgG antibodies, which provide the majority of antibody-based immunity. Analysis of sera collected at 4 weeks revealed that mice immunized intranasally with either vaccine had significantly higher levels of spike protein-specific IgG than did mice that were immunized intramuscularly, and these high levels were sustained through 8 weeks post-vaccination. However, only lung wash samples from mice immunized with either vaccine intranasally contained detectable levels of spike protein-specific IgG at the 4 week time point, suggesting that only intranasal immunization led to IgG production in the airways. Interestingly, the airways of Tri:ChAd recipients harbored almost twice as much spike-specific IgG as those of Tri:HuAd recipients, a difference that was not observed in the serum, suggesting that the vector of chimpanzee origin induced better local immune responses when administered via the nose. Additionally, only the lung wash samples from intranasal Tri:ChAd recipients contained any observable anti-spike IgA antibodies, which are prevalent in mucosal surfaces and represent a first line of defense against invasion by inhaled and ingested pathogens. These results suggested that both vaccines, but Tri:ChAd in particular, induced better immune responses when they were delivered intranasally.

Lung wash samples were also analyzed for vaccine antigen-specific T cells by detecting characteristic cell surface proteins on these T cells, and for specific cytokines produced by the T cells in response to their exposure to peptides encoding each of the three vaccine-encoded antigens: S1, nucleocapsid, or RdRp. This analysis revealed that when the vaccine was injected into the muscle, neither Tri:HuAd nor Tri:ChAd induced detectable airway antigen-specific cytotoxic T cells, which are capable of killing virus-infected cells. In contrast, both vaccines induced significant numbers of cytotoxic T cells in the airways when they were delivered intranasally. Although the detected T cells were specific for all three antigens, S1-specific cells were more abundant, and they were also multifunctional in that they produced more than one cytokine characteristic of cytotoxicity. However, in the context of intranasal vaccination, the Tri:ChAd vaccine induced higher total numbers of cytotoxic T cells than the Tri:HuAd vaccine, suggesting that a single dose of Tri:ChAd delivered intranasally is highly effective at inducing vaccine antigen-specific cytotoxic T cells in the respiratory tract.   

Interactions between lung macrophage cells and SARS-CoV-2 can increase the expression of the host-derived peptide MHCII on macrophage surfaces, allowing these cells to become a critical component of “trained innate immunity.” This altered innate immunity results from a functional modification of innate immune cells that leads to an antibody-independent temporary immune memory that is characterized by an accelerated response to a second challenge with the same pathogen. Neither Tri:HuAd nor Tri:ChAd induced detectable MHCII expression on macrophages in the lungs of mice when delivered intramuscularly. In contrast, both vaccines induced increased levels of MHCII-expressing lung macrophages 8 weeks after immunization when delivered intranasally, with a higher number of these cells in the lungs of Tri:ChAd recipients, suggesting that intranasal immunization with Tri:ChAd induces strong trained innate immunity.

The authors next tested the ability of the vaccines to protect mice from SARS-CoV-2 infection by intentionally infecting immunized mice with a lethal dose of SARS-CoV-2 at 4 weeks after immunization, and monitoring them for clinical signs of illness in terms of weight loss. Unvaccinated control animals and 80% of animals immunized intramuscularly with either vaccine were not protected from infection or disease and died by 5 days post-infection. In contrast, animals immunized with Tri:HuAd showed a temporary and slight weight loss, but rebounded over the course of two weeks, while animals immunized with Tri:ChAd did not lose weight at all for the duration of the two-week time course. This result correlated with reduced viral load and reduced lung pathology at 14 days post-infection in mice immunized intranasally compared to unvaccinated controls and intramuscular vaccine recipients, with Tri:ChAd recipients exhibiting the most reduced viral burden and lung pathology.   

Having determined that intranasal administration of Tri:ChAd induced the best immune responses, all subsequent experiments were carried out using this vaccination strategy. To assess the individual contributions of B cells and T cells in the vaccine-induced immune response, the authors immunized two groups of mice: one group that was genetically deficient in B cells and a second group of genetically wild type mice that were depleted of T cells by receiving injections of antibodies against T cell-characteristic cell surface molecules. Because T cells are needed for B cell stimulation and production of antibodies, the antibody injections were done on day 25 after immunization to ensure that vaccine-induced B cell functions and antibody production remained intact in the T cell-depleted mice. At 28 days after vaccination, both groups of animals were infected with SARS-CoV-2 and monitored for weight loss. Unvaccinated mice that were either B cell-deficient or T cell-depleted and control unvaccinated mice with normal B and T cell functions were subject to drastic weight loss. In stark contrast, neither B cell-deficient nor T cell-depleted mice that had been intranasally immunized with Tri:ChAd showed any weight loss throughout the 2-week experimental time course, despite the fact that both groups of animals had significantly elevated viral burden in the lung. This result suggested that Tri:ChAd was immunogenic enough to compensate for the lack of B or T cells and protect the mice from clinical disease after SARS-CoV-2 infection.

The authors next wanted to analyze the role of trained innate immunity in the absence of B and T cell immunity, so they immunized two groups of mice. The first group consisted of B cell-deficient animals that were depleted of T cells shortly after vaccination. Because T cells induce vaccine-mediated trained innate immunity early after vaccination, this strategy produced mice that lacked B cells, T cells, and trained innate immunity. The second group consisted of B cell-deficient animals that were depleted of T cells starting at day 25 post-vaccination to allow for induction of trained innate immunity and thus produce mice that lacked B cells and T cells, but possessed intact trained innate immunity. At 28 days after vaccination, both groups of animals were infected with SARS-CoV-2 and monitored for weight loss and lung pathology. Unvaccinated control mice with functional B cells, T cells, and trained innate immunity and unvaccinated mice lacking B cells, T cells, and trained innate immunity showed severe weight loss and severe lung pathology. In contrast, vaccinated mice lacking B cells and T cells but possessing trained innate immunity appeared healthy with no weight loss and no pathology in the lungs, suggesting that the immune protection observed in animals lacking B cells, T cells, or both, was likely mediated by trained innate immunity.

The study demonstrated similar results after infection of mice with alpha and beta variants of SARS-CoV-2; however, it would be interesting to see if these vaccines have similar efficacy against the later variants, including delta and omicron. Although these results are promising, it is important to remember that mice can be poor predictors of human disease outcomes. Nevertheless, I look forward to seeing the results of the clinical trial that is currently underway to compare the relative efficacies of Tri:HuAd and Tri:ChAd following intranasal delivery in humans.

[For a more detailed discussion of this paper, please check out TWiV 867.]

Spikevax Induces Durable Protection from the Delta Variant in Rhesus Macaques

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by Gertrud U. Rey

It is currently not clear how long SARS-CoV-2 vaccine-induced immunity lasts. The gold standard for determining the efficacy of a vaccine is the “challenge” study, which involves intentionally infecting immunized subjects with the pathogen against which they were immunized. Such studies are typically done in non-human primates, because it is unethical to deliberately infect humans with pathogens that cause serious morbidity and mortality.

A recent preprint by Kizzmekia Corbett and others describes experiments done to assess the efficacy of Moderna’s SARS-CoV-2 “Spikevax” vaccine in rhesus macaques one year after vaccination. The authors immunized eight animals with two doses of Spikevax at four-week intervals and then collected blood samples, nasal swabs, and lung wash samples at various time points over the course of the following year. The macaques were then challenged with the SARS-CoV-2 delta variant virus at 49 weeks, and more samples were collected at different time points after challenge.

Blood samples collected at 6, 24, and 48 weeks post-vaccination were used to analyze the ability of IgG antibodies in these samples to bind the receptor-binding domains of three different viruses: 1) “ancestral” SARS-CoV-2, which had the exact spike protein antigen encoded in the vaccine, 2) the delta variant, and 3) the beta variant. These latter two had variant spike proteins. IgG antibodies are mostly blood-resident and provide the majority of antibody-based immunity against invading pathogens. IgG levels were highest at 6 weeks after vaccination for all three viruses; they then declined rapidly between 6 weeks and 24 weeks, and more slowly between 24 weeks and 48 weeks. Most IgG detected at 6 weeks bound ancestral virus, with 5.4-fold and 8-fold fewer IgG molecules binding the delta and beta variants, respectively. However, when delta and beta variant-specific IgG antibodies were tested for their ability to block binding between SARS-CoV-2 and its cognate ACE2 receptor, they inhibited almost 100% of binding of both delta and beta variant viruses, suggesting that the antibodies were still functional in preventing infection, in spite of their diminished quantity.

The ability of blood-resident IgG antibodies to neutralize the three respective viruses followed a similar trend, with a gradual decline in neutralizing activity against all viruses by 48 weeks post-vaccination. Interestingly, even though the quantity of total binding and neutralizing antibodies targeting the delta variant decreased over time, the number of antibodies targeting regions associated with neutralization increased. In addition, the binding avidity of antibodies to ancestral virus increased significantly between week 6 and 24 and remained steady through week 48 post-vaccination. In contrast to affinity, which measures the strength of the binding interaction between antigen and antibody at a single binding site, avidity measures the total binding strength of an antibody at every binding site. These two shifts – the increase in the number of antibodies binding targets associated with neutralization and the increase in antibody avidity over time in spite of a decrease in total antibody levels – are suggestive of a maturing immune response that is more focused on viral regions of high immunological relevance. It is noteworthy to mention that the regions associated with neutralization are outside of areas where the variant viruses have accumulated changes in the spike protein, further implying that Spikevax and other SARS-CoV-2 vaccines are just as effective against viral variants as they are against ancestral virus.

Next, the authors analyzed lung wash samples and nasal swabs for delta-binding IgG and IgA antibodies. IgA antibodies are predominantly found in mucus membranes and their fluids, where they protect against invasion by inhaled and ingested pathogens. IgG kinetics in the lung were similar to those observed in the blood – both binding and neutralizing IgG to all three viruses were highest at 6 weeks after vaccination and decreased steadily over time until they were indistinguishable to those observed in unvaccinated animals. In contrast, IgG levels in the nose increased through week 25, plateaued, and remained stable through week 42 post-vaccination. IgA levels in the lung were highest at week 6 post-vaccination, but decreased to levels similar to those observed in unvaccinated animals by week 24. IgA levels in the nose were similar to those in unvaccinated animals at all time points. These results suggest that although SARS-CoV-2 vaccination may not induce a detectable mucosal immune response in the nose, it does induce good initial mucosal immunity in the lung, which is typically the site of severe COVID-19. This immunological difference between the lung and the nose might also explain why SARS-CoV-2 vaccines are more effective at preventing severe disease than at preventing infection.

The authors also analyzed blood samples from vaccinated animals for the presence of SARS-CoV-2-specific memory B cells, which can quickly produce spike-specific antibodies upon subsequent exposure to SARS-CoV-2. At week 6 post-vaccination, about 0.14% of all memory B cells were specific for the ancestral virus, and about 0.09% were specific for the delta variant. In comparison, about 2.5% of all memory B cells were specific for both the ancestral virus and the delta variant, and this high proportion of dual-binding to single-binding cells remained constant through week 42 post-vaccination.

To see whether these vaccine-induced immune parameters are protective after viral challenge, the authors infected the animals with delta variant virus at 49 weeks after the initial immunization. Lung washes and nasal swabs were collected on days 2, 4, 7, and 14 after challenge to monitor viral replication. On day 2 after challenge, vaccinated animals had about 11-fold fewer viral RNA copies per milliliter in their lungs than unvaccinated animals, and these RNA levels declined rapidly over the following days. In contrast, viral RNA levels in unvaccinated animals remained significantly elevated through day 7 post-infection. Viral RNA levels in the nose followed a similar trend; however, their decline in vaccinated animals was not as dramatic as that observed in the lung.

Antibodies to all three viruses in the lungs of vaccinated animals were significantly higher on day 4 after challenge than at week 42 after immunization, suggesting that memory B cell responses to infection were quick and robust. Viral challenge after vaccination also induced both T helper cells, which stimulate B cells to make antibodies, and cytotoxic T cells, which kill virus-infected cells. Analysis of lung tissue also revealed that vaccination prevented lung pathology and protected the lower respiratory tract from severe inflammation after infection.

Perhaps the most interesting observation in the study relates to whether vaccinated individuals who become infected replicate and transmit virus to others. When the authors analyzed lung wash samples for T cells specific for the SARS-CoV-2 N protein, which is not encoded in the Spikevax vaccine, they only found these cells in unvaccinated animals. This suggests that even though it had been one year since vaccination, immunized animals that were then infected did not replicate the challenge virus to a sufficient extent to produce T cells specific for the SARS-CoV-2 N protein – a response that would only be elicited by actual infection with whole virus. In other words, the memory response to the SARS-CoV-2 spike protein induced by the vaccine eliminated incoming virus so quickly that the immune system had no chance to mount a response to the viral N protein encoded in the challenge virus, presumably because the virus was cleared quickly.

In summary, vaccinated animals appear to be better protected from severe disease and to clear virus faster than unvaccinated animals. This result aligns with data published in a previous preprint, which showed that viral RNA levels in delta variant-infected people who had been vaccinated prior to infection declined more rapidly than in people who were not vaccinated. And although monkeys are not human, previous studies assessing the protective efficacy of Spikevax have shown that rhesus macaques are reliably predictive of outcomes in humans, making them a great model for determining the effects of waning antibody levels on long-term protection against SARS-CoV-2 infection.

[Kizzmekia Corbett, a viral immunologist at Harvard who was central to the development of the Moderna mRNA vaccine, was previously a guest on TWiV 670. The preprint described in this post was also discussed on TWiV 824.]

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.]

Are the SARS-CoV-2 Vaccines Safe for Pregnant and Lactating People?

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by Gertrud U. Rey

Vaccination is the gold standard for preventing infectious diseases and reducing the impact of emerging pathogens. As more and more people are becoming immunized against SARS-CoV-2, a prominent question continues to arise: are the vaccines safe for pregnant and breast-feeding people?

(Image credit: Shutterstock)

None of the clinical trials for SARS-CoV-2 vaccines included pregnant or lactating subjects. Pregnant people in particular are a historically difficult group to study in the context of drug safety because of concerns of liability over potential adverse effects of a new product on a fetus. This lack of safety data further complicates treatment of SARS-CoV-2 infection in pregnant people, who already have an increased risk for premature birth and miscarriage if they become infected. Based on these risk factors, the currently available data, and interim recommendations by the Advisory Committee on Immunization Practices for people over the age of 16, the American College of Obstetricians and Gynecologists recommends that pregnant and lactating people should have access to SARS-CoV-2 vaccines. Fortunately, many pregnant and lactating people have opted to be vaccinated, and the data on the safety and efficacy of vaccination in these two cohorts are beginning to emerge.

A recent publication in the American Journal of Obstetrics and Gynecology describes the safety and immunogenicity of SARS-CoV-2 vaccines in pregnant and lactating women compared to non-pregnant controls and women who had been naturally infected with SARS-CoV-2 during pregnancy. The researchers collected blood and breast milk from 131 women of reproductive age, including 84 pregnant, 31 lactating, and 16 non-pregnant participants who had received two doses of either the Pfizer/BioNTech or Moderna mRNA COVID-19 vaccine; and analyzed the samples for the presence of IgG and IgA antibodies. IgG antibodies are mostly present in the blood and provide the majority of antibody-based immunity against invading pathogens. IgA antibodies are predominantly found in mucus membranes and the fluids secreted by these membranes, including saliva, tears, and breast milk. IgA antibodies are particularly suitable for protecting against pathogens like SARS-CoV-2, which invade the body through these membranes.

Samples were collected at the time of the first and second vaccination, 2 to 6 weeks after the second dose, and at delivery. The researchers also collected umbilical cord blood from 10 infants born during the study period.

The main findings were as follows. Side effects resulting from vaccination were rare and occurred with similar frequency in all participants. These findings are consistent with post-vaccination data tracked by the CDC via the V-safe application, suggesting that post-vaccination reactions in pregnant people are not significantly different from those in non-pregnant people.

Pregnant, lactating, and non-pregnant vaccine recipients produced significantly higher levels of SARS-CoV-2-specific IgG than pregnant women who had been naturally infected. Interestingly, the second vaccine dose led to increased concentrations of coronavirus-specific IgG but not IgA, in both maternal blood and breast milk. The authors speculate that this lack of IgA boosting may have to do with the intramuscular route of immunization, which usually does not induce very high levels of serum IgA. 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. In contrast, natural infection induces higher levels of mucosal (IgA) antibodies because SARS-CoV-2 enters the body through the mucosal surfaces. Nevertheless, studies have shown that IgG in breast milk is critical for protecting newborns from other pathogens like HIV, RSV, and influenza. It is possible that this type of antibody is equally important for protecting infants from SARS-CoV-2.

It was previously unclear whether vaccine-induced SARS-CoV-2 antibodies are transferred across the placenta to the fetus. However, the authors also found vaccine-induced IgG in cord blood, suggesting that these antibodies do cross the placenta, as has been previously observed after vaccination of pregnant people against influenza virus, Bordetella pertussis, and other pathogens. Whether these antibodies protect the baby from SARS-CoV-2 infection after birth, remains to be determined.

This study provides the first set of clinical data suggesting that vaccination of pregnant and lactating people against SARS-CoV-2 is both safe and beneficial. Some people have expressed concern over the possible incidence of fever following the second vaccine dose in pregnant people; however, the potential risk to the fetus from a 24-hour fever must be weighed against the possibility of chronic, severe COVID-19 in the mother. Accumulating data suggest that the benefits of vaccination during pregnancy and lactation outweigh the risks of adverse effects to the fetus or infant and align with the abundance of clinical data showing the beneficial effects of vaccinating these groups of people against influenza virus. Analyses of cord blood from infants whose mothers were naturally infected with SARS-CoV-2 during pregnancy indicate that the potential for transfer of antibodies is greater early during gestation, suggesting that immunization during the earlier stages of pregnancy might be preferable. However, more studies are needed to assess the optimal timing of maternal vaccination to achieve enhanced neonatal immunity. In the meantime, these preliminary data allow pregnant and lactating people to make more informed decisions, and also aid physicians in providing evidence-based recommendations.

My at-home SARS-CoV-2 antibody test result

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Recently I had a false positive PCR result for SARS-CoV-2. To confirm that I was not infected, I did an assay for viral antibodies in my blood using a rapid, at home, lateral flow assay. In this video I explain how the assay works and what my results mean.

The Route Matters

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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.