virology blog

About viruses and viral disease

Gertrud Rey

RNA, in a Nutshell

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by Gertrud U. Rey

It is now a little more than a year since the emergence of SARS-CoV-2, and we already have several highly effective vaccines against this virus. Because of my previous research experience in vaccine science, I was very skeptical about the promise of a SARS-CoV-2 vaccine this soon. I was wrong, and I could not be happier about that.

Two of the leading vaccines were developed by Pfizer/BioNTech and Moderna and consist of a messenger RNA (mRNA) that encodes the full-length SARS-CoV-2 spike protein. Upon injection into a vaccine recipient, the mRNA would enter cells and be translated by the host protein synthesis machinery into the SARS-CoV-2 spike protein, which would then serve as an antigen to promote an immune response. mRNA vaccines are non-infectious and do not integrate into the genome, meaning that there is no risk of infection or mutations caused by inserted vaccine sequences. Although these vaccines are the first of their kind to be licensed for widespread use, the concept is not new. Reports of the first successful translation of a foreign mRNA in animals were published in 1990, and this technology has been refined ever since. Progress in the field has been hampered by concerns that the inherent instability of RNA would prevent its use for delivery as a therapeutic or vaccine. However, research has shown that the stability of RNA can be increased through various modifications and delivery methods.

One way a vaccine mRNA molecule can be modified is by placing it between two RNA sequences that don’t code for protein, i.e., untranslated regions (UTRs; see graphic), which stabilize the mRNA and optimize it for translation. The ends of the mRNA – also known as the 5′ and 3′ ends, respectively – can be further modified by addition of a “cap” and a “poly(A) tail.” The cap consists of a modified guanosine nucleotide followed by three phosphates (“G-PPP” in the graphic) and serves as a recognition signal for the cellular ribosome to bind and translate the mRNA. The poly(A) tail is a string of adenosine nucleotides (“AAA” in the graphic), which further stabilize the mRNA.

A common method for encapsulating and delivering the mRNA into cells is to encase it in a cocoon of phospholipids. For example, the mRNA molecule in both the Pfizer and Moderna vaccines is encapsulated in a lipid nanoparticle (pictured), which protects the mRNA from degradation and ensures proper delivery into cells. Addition of cholesterol molecules makes the nanoparticle more fluid and is thought to increase its ability to fuse with our cell’s membranes to deliver the mRNA into our cells. Addition of polyethylene glycol (PEG) increases the potency of the vaccine particle by hiding it from the host immune system, making it more water soluble, and slowing its degradation.

One of the reasons why SARS-CoV-2 mRNA vaccines could be produced so quickly is because all this basic science was already in place at the start of the pandemic. And although the SARS-CoV-2 vaccines are the first mRNA vaccines to be authorized by the FDA for emergency use, several mRNA vaccines have undergone clinical trials in humans before, for at least four infectious diseases: rabies, influenza, cytomegalovirus infection, and Zika virus infection.

Another factor that helped speed up the process of SARS-CoV-2 vaccine production is that, luckily, scientists were able to extrapolate the insight gained from the study of other coronaviruses to SARS-CoV-2. Like the spike protein of other coronaviruses, the SARS-CoV-2 spike protein is highly immunogenic and is targeted by neutralizing antibodies, which bind viral antigens to inactivate the virus and prevent infection of new cells. The spike protein also mediates binding of the virus to the ACE2 host cell receptor via spike’s receptor-binding domain and fusion of the viral particle with the host cell membrane via spike’s fusion domain. However, to mediate this fusion, the SARS-CoV-2 spike protein undergoes a structural rearrangement from its pre-fusion conformation. By 2017, scientists at the Vaccine Research Center of the National Institute of Allergy and Infectious Diseases had already determined that the pre-fusion form of Middle East respiratory syndrome coronavirus (MERS-CoV) is more immunogenic than its post-fusion form. Accordingly, they had spent several years engineering a mutation that locks the translated spike protein into its pre-fusion structure. When the SARS-CoV-2 genome sequence was published one year ago, scientists were able to compare it to the MERS-CoV sequence and identify the exact location where the pre-fusion stabilizing mutation had to be made. And luckily, making the mutation in the SARS-CoV-2 spike mRNA sequence stabilized the spike protein in its pre-fusion conformation. 

Conventional vaccine strategies have repeatedly failed to yield vaccines against challenging viruses like HIV-1, herpes simplex virus, and respiratory syncytial virus (RSV), while recent advances in mRNA vaccine technology show promise in immunizing against some of these viruses. For example, RSV poses a substantial public health threat due to its association with severe morbidity and mortality in infants and premature babies. Despite 60 years of continual efforts, we still don’t have a licensed RSV vaccine, in part because natural RSV infection does not induce a durable immune response. We do know that the RSV F (fusion) protein is highly conserved and elicits broadly neutralizing antibodies, and recent studies have shown that similar to the case of the SARS-CoV-2 spike protein, most neutralizing activity in human serum is directed against the pre-fusion form of the RSV F protein. This observation inspired scientists at Moderna to develop an RSV pre-fusion F protein mRNA vaccine, with Phase I clinical trial data showing promising results.

One of the exciting features of the mRNA vaccine platform is that it is not only applicable to preventing viral diseases but can also be used for treating cancer. Cancer mRNA vaccines would target tumor-associated antigens that are preferentially expressed in cancer cells.

Vaccination remains one of the most effective public health measures for preventing and controlling infectious diseases. However, conventional vaccine approaches using live-attenuated and inactivated virus vaccines are time-consuming and expensive. mRNA vaccines can be produced more quickly and cost-effectively than conventional vaccines because they obviate the need for growing and/or repeatedly passaging viruses in cell culture. Nonetheless, we would not know any of this without decades of prior studies, which further highlights the importance of regularly funding basic research.

Pandoraviruses Are Not Alive

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by Gertrud U. Rey

Viruses are universally defined as “obligate intracellular parasites” because they cannot replicate outside of a host cell and depend on that cell and its various metabolic factors for replicating their genome. Based on this definition, most virologists agree that viruses are not alive. 

When giant viruses were initially discovered, they were found to violate multiple principles of virology. For example, mimiviruses can be parasitized by small viruses called virophages that can only replicate if they confiscate the replication factors of a co-infecting mimivirus. Because the virophage also inactivates the mimivirus during this process, some interpret this scenario as a virus infecting another virus, a previously unheard-of phenomenon. In turn, mimiviruses have defense mechanisms that inhibit virophage replication, a property that is analogous to eukaryotic anti-viral interferon-mediated defenses. Additionally, mimiviruses encode proteins that participate in protein synthesis – another unusual property for a virus.

Some mimiviruses also have a gene that codes for citrate synthase, an enzyme involved in the Krebs cycle. The Krebs cycle is integral to cellular metabolism in living organisms because it ultimately powers the production of adenosine triphosphate (ATP), the cell’s molecular currency of energy. The cycle takes place in the matrix of the mitochondrion (pictured), where it feeds electrons into a string of complexes in the inner mitochondrial membrane known as an electron transport chain. As electrons move down this chain, they release energy, which is used by membrane-resident enzymes to pump protons from the matrix across the membrane into the intermembrane space (pictured as “proton pump”). This produces a concentration gradient, which is a difference in the concentration of protons on one side of the membrane compared to the other. To achieve equilibrium, the protons move back into the matrix through the action of another membrane resident enzyme called ATP synthase, which captures the energy of the protons to produce ATP. In other words, a concentration gradient across a membrane produces an electrical potential and is usually associated with the ability to generate energy in living cells.    

Based on the knowledge that some mimiviruses encode a component of the Krebs cycle, a group in Marseille wanted to determine whether giant viruses can produce their own energy. To do this, they infected a species of amoeba, the natural host of giant viruses, with Pandoravirus massiliensis, a virus with the largest known viral genome encoding many proteins with unknown functions. 

The authors isolated viral particles from P. massiliensis-infected amoebae and treated them with P. massiliensis-specific antibodies and a dye that detects electrical potential. This technique produced fluorescence in the membranes of P. massiliensis particles, indicating the presence of an electrical potential, in contrast to control virus particles isolated from cells infected with cowpoxvirus, which did not fluoresce. To confirm that the observed fluorescence represented a real concentration gradient with potential for electron transport, the authors treated the P. massiliensis particles with CCCP, a chemical that inhibits movement of electrons. This treatment led to diminished membrane fluorescence, suggesting that the observed membrane potential was real. Interestingly, the intensity of the electrical potential could be modified with addition of variable concentrations of acetyl-CoA, a known regulator of the Krebs cycle. 

In an effort to determine how the P. massiliensis genome could play a role in energy metabolism, the authors did a sequence alignment with a database of conserved sequence domains known to be involved in energy metabolism. This revealed that P. massiliensis contains genes for nearly all enzymes in the Krebs cycle, but when these genes were cloned and expressed in bacterial cells, only one of them, isocitrate dehydrogenase, was functional. In agreement with this observation, the authors also found that mature P. massiliensis particles released from amoeba cells did not produce any ATP. Nevertheless, when amoeba cells were infected with P. massiliensis that were pre-treated with CCCP, they produced a lower number of viral particles, suggesting that the observed membrane potential might play a role during infection. 

The authors conclude that these findings “position this virus as a form of life.” I disagree with this conclusion for the following reasons. Although P. massiliensis encodes numerous Krebs cycle enzymes, only one of them seems to be functional. Furthermore, P. massiliensis particles did not produce any ATP, meaning that this virus cannot produce its own energy. Even if it did, it still depends on the host cell for many other replication factors, including those needed to make proteins. As long as a virus requires a cell for replication, it is still a virus, and hence not alive.

Still, these findings are interesting and remind me of bacteriophage ϕKZ, a giant virus discussed in a previous post. After infecting a bacterial cell, ϕKZ assembles a nucleus-like shell, which shields the viral DNA from bacterial immune enzymes. Any discovery that reveals genes in viruses that suggest the potential for cell-like functions raises at least a couple of questions. Are these genes remnants of cellular genes, thereby suggesting that these viruses originated from ancient parasitic cells? Or did these giant viruses acquire the genes over time to gain more independence from host cells? Either way, pandoraviruses are aptly named because their study continues to yield surprising discoveries.

T Cell Responses to Coronavirus Infection are Complicated

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by Gertrud U. Rey

Throughout the current pandemic, there has been a lot of talk about T cells and their role in protecting against SARS-CoV-2 infection and disease. Some data suggest that 20-50% of people with no prior exposure to SARS-CoV-2 have T cells that recognize SARS-CoV-2 peptides, and that these T cells may be a result of recent infections with one or more of the seasonal human coronaviruses. However, it is unclear whether these “cross-reactive” T cells actually protect from SARS-CoV-2 infection and disease.  

T cells are an important part of the adaptive immune response, which initiates during a first exposure to a pathogen and protects from re-infection and disease upon a second exposure to the same pathogen. During that first exposure, T helper cells sense the presence of one or more proteins (i.e., 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, white blood cells to destroy ingested microbes, and cytotoxic T cells to directly kill infected target cells (see schematic). Before T cells encounter their first antigen, they are considered to be “naïve.” Upon their first contact with an antigen, they begin to mature and differentiate into either cytotoxic T cells or memory T cells. As we age and encounter more and more pathogens, the ratio of our memory T cells to our naïve T cells increases – a phenomenon sometimes referred to as “immunological age.” 

Based on evidence from several labs, some have suggested that pre-existing cross-reactive memory T cells in people with no prior exposure to SARS-CoV-2 may have a protective effect. However, recent findings indicate that this may not be the case.

Several research groups from the U.S. and Australia analyzed blood samples from individuals with no prior SARS-CoV-2 exposure with the intent of better defining the range of T helper cells that can recognize antigenic portions known as epitopes in the SARS-CoV-2 genome. To ensure that the blood donors had never been infected with SARS-CoV-2, the researchers used stored samples that had been collected between 2015 and 2018. The authors found that the blood samples contained T cells that can recognize SARS-CoV-2 sequences that have at least 67% similarity to seasonal coronavirus sequences. However, the authors also found that people who had experienced a previous infection with SARS-CoV-2 had stronger and more specific (i.e., higher avidity) memory T cell responses to SARS-CoV-2 peptides than people with no prior exposure, and more than half of these responses were directed to epitopes in the spike protein. This suggests that SARS-CoV-2 memory T helper cells preferentially target viral proteins that are made in abundance during infection. 

The authors conclude that an infection with a seasonal coronavirus may induce a range of memory T cells that have substantial cross-reactivity to SARS-CoV-2. However, they are careful to note that the clinical relevance of these data remains unclear and that there is no evidence that these memory T cells have any functional role in protecting from infection or disease.  

Based on these findings, several groups of investigators in Germany wanted to determine whether the observed pre-existing T cell memory response in people with no prior SARS-CoV-2 exposure is protective against COVID-19. They first tested T cell activity in the blood of donors with and without prior exposure to SARS-CoV-2 by exposing their blood to highly immunogenic SARS-CoV-2 peptides. Blood from donors with a prior exposure contained memory T cells that recognized SARS-CoV-2 peptides very well, especially peptides derived from the spike, membrane, and nucleocapsid proteins. Blood from donors with no prior exposure also contained low levels of SARS-CoV-2-reactive memory T cells, but this reaction was more scattered and directed against multiple viral proteins. 

To further characterize the pre-existing cross-reactive T cells in blood samples from people with no prior SARS-CoV-2 exposure, the authors compared the numbers of memory T cells and naïve T cells in the blood samples by analyzing them for the presence of protein markers that are characteristic for each type of cell. A substantial portion of SARS-CoV-2-reactive T cells from donors with no prior exposure were naïve T cells, whereas from COVID-19 patients most were mature memory T cells. Because memory T cells are more easily activated following an infection than naïve T cells, the authors speculate that deficient, low avidity memory T cells in people with no prior SARS-CoV-2 exposure may compete with naïve T cells and prevent their activation and maturation into highly specific memory T cells upon infection with SARS-CoV-2. This could potentially lead to an inferior immune response in people with no prior SARS-CoV-2 exposure. 

Some have suggested that young patients and children may be particularly well protected from SARS-CoV-2 infection and/or disease because they are frequently infected with seasonal coronaviruses and thus presumably have high levels of pre-existing memory T cells. However, the authors found that compared to young people, older people with no prior SARS-CoV-2 exposure actually had higher numbers of SARS-CoV-2 cross-reactive memory T cells, but these T cells had a decreased avidity to SARS-CoV-2 epitopes compared to memory T cells from people with prior SARS-CoV-2 exposure.   

A further comparison of T cell responses in patients with mild or severe COVID-19 revealed that although the latter had high numbers of SARS-CoV-2 specific T cells, these T cells had reduced target specificity and avidity compared to T cells from patients with moderate disease. The authors conclude that this unfocused response may result from recruitment of a broad range of pre-existing memory T cells in people with increased immunological age and may contribute to development of severe COVID-19 in the elderly. 

The initial discovery of SARS-CoV-2-specific memory T cells in individuals with no prior exposure to SARS-CoV-2 had inspired the hypothesis that these T cells could possibly protect these individuals from disease and might partially explain why children are less susceptible to COVID-19. However, in light of these new findings it is likely that pre-existing SARS-CoV-2-specific memory T cells may contribute to the wide spectrum of disease severity among the general population and may actually be partially responsible for severe COVID-19 in the elderly. 

[For an in-depth discussion of these two papers, I recommend TWiV 657 and Christian Drosten’s “Das Coronavirus Update,” episodes 58 and 60.]

You Don’t Need the Whole Antibody

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by Gertrud U. Rey

Antibodies are large proteins that are made by B cells of the adaptive immune system. Most people think that antibodies function only as a whole molecule, but some of the individual fragments of an antibody can also bind and neutralize antigens. 

An antibody consists of two heavy chains and two light chains that assemble into a Y-shaped structure (left side of Figure). The stem of the Y is known as the “fragment crystallizable” (Fc) portion and is composed of two heavy chains. The two arms of the Y are known as the “fragment antigen-binding” (Fab) portions and are each composed of one heavy chain and one light chain. As its name suggests, the top half of each Fab fragment is the antigen-binding region of the antibody, and it is variable – meaning that it varies between antibodies that are produced by different B cells. The bottom half of each Fab fragment and the entire Fc region are constant, meaning that they are identical in all antibodies of the same isotype, but differ in antibodies of different isotypes. For example, the constant regions are identical in all IgG antibodies but differ between IgG and IgA antibodies. 

In an effort to identify anti-SARS-CoV-2 antibodies suitable for preventing and treating SARS-CoV-2 infection, the authors of a recent publication screened 100 billion different anti-SARS-CoV-2 antibody candidates for their ability to bind and/or neutralize SARS-CoV-2. This eventually led to the discovery of “ab8,” an antibody fragment consisting of a variable heavy (VH) region and having particularly potent SARS-CoV-2 binding specificity and neutralization activity. To increase the binding avidity of ab8 (i.e., the stability of its interaction with an antigen) and extend its longevity in the human body, the authors fused this fragment to the Fc domain of human IgG1, an abundant and stable type of human antibody. This produced the molecule hereinafter referred to as “VH-Fc ab8″ (right side of Figure).  

The authors found that VH-Fc ab8 can bind various conformations of the SARS-CoV-2 spike protein, including when the spike protein is bound to a cell surface. VH-Fc ab8 can also bind to and neutralize six different SARS-CoV-2 isolates having different amino acid changes in the receptor-binding domain, suggesting that it is broadly cross-reactive. Notably, it does not bind to human cells, meaning that it does not seem to interfere with normal cellular functions.

As a next step, the authors evaluated the ability of VH-Fc ab8 to prevent SARS-CoV-2 infection in mice. If given to mice before they were infected with SARS-CoV-2, VH-Fc ab8 inhibited viral replication at all doses tested, but it only neutralized virus at the highest dose of 36 mg/kg. Although these results were encouraging, it is often difficult to interpret data obtained in mice in terms of clinical relevance in humans, because mice don’t develop the COVID-19-related disease pathologies observed in humans. Hamsters more closely imitate human SARS-CoV-2 infection in the lung, suggesting that they could be a useful mammalian model for COVID-19. VH-Fc ab8 caused significantly reduced levels of infectious virus in the lung, nasal mucosa, and saliva of hamsters when administered one day before (i.e., “prophylactically”) or six hours after SARS-CoV-2 infection (i.e., “therapeutically”) compared to untreated control animals, suggesting that it could be used to both prevent and treat SARS-CoV-2 infection. Although VH-Fc ab8 led to greater reduction of virus levels when given prophylactically than when given therapeutically, therapeutic administration still led to significantly decreased viral loads in treated animals compared to untreated control animals, even at very low doses. VH-Fc ab8 not only alleviated pneumonia and reduced lung viral loads in hamsters, but it also reduced virus shedding in the upper airway, which could help with reducing transmission. 

The authors also found that when they gave hamsters the same dose of either VH-Fc ab8 or IgG1 ab1 – a full-sized version of the antibody, and then examined their concentrations in the serum five days later, levels of VH-Fc ab8 were significantly higher than those of the full-sized antibody. This suggests that the systemic distribution of VH-Fc ab8 is more long-lived than that of a full-sized antibody. 

Although small animal models can provide key insights into the pathogenic mechanisms of viral infections, they are often poor predictors of human disease outcomes. The therapeutic timeline followed in the hamster experiments (i.e., administration of VH-Fc ab8 six hours after infection) would also be difficult to reproduce in humans because therapeutic drugs are not usually administered until well after symptom onset. Therefore, it would be difficult to determine whether the therapeutic effect of VH-Fc ab8 observed in hamsters would be the same in humans. 

That being said, there are clear advantages to using antibody fragments instead of whole antibodies. Their small size allows them to penetrate more efficiently to sites of infection and bind antigens more easily and with more specificity. Smaller molecules also diffuse more easily through tissues, meaning that they could be administered by routes other than injection, such as by inhalation. Furthermore, because the molecular weight of VH-Fc ab8 is only about half that of a full-sized antibody, smaller quantities would be needed to obtain the same number of molecules, meaning that antibody fragment therapeutics could be more easily mass-produced. 

There is no question that we are in dire need of an effective therapeutic drug to treat SARS-CoV-2 infection. If the results observed in these animal experiments can be duplicated in humans, VH-Fc ab8 would be an attractive option for both treating and preventing SARS-CoV-2 infection. 

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. 

How to End this Pandemic

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by Gertrud U. Rey

As of today, SARS-CoV-2 has infected 18.7 million people and caused 700,000 deaths worldwide. The most realistic way to quickly curb the spread of the virus would require daily identification and isolation of individuals who are contagious, a process that is hampered by cumbersome sampling and testing methods with slow turnaround times. 

The predominant test for diagnosing SARS-CoV-2 infection is a highly sensitive assay called quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). To carry out a SARS-CoV-2 qRT-PCR test, a mucus sample is processed to inactivate virus particles and extract the viral RNA. The RNA is converted to DNA (the reverse transcription step), which is then amplified during the polymerase chain reaction portion of the assay. For amplification to occur, a small piece of DNA (a primer) binds to a complementary target sequence in the SARS-CoV-2 DNA, while another piece of DNA (a probe) attaches to a sequence downstream of the primer binding site. Binding of the primer initiates amplification of the target DNA by an enzyme called polymerase, which copies the DNA in one direction towards the probe. Once the polymerase reaches the probe, it cleaves it, which activates a fluorescent marker attached to the probe. The use of this fluorescent probe allows for monitoring of the fluorescent signal quantitatively in real time rather than just detecting an accumulated end product. 

While the qRT-PCR test is very sensitive, it also has multiple limitations. It requires expensive laboratory instrumentation and trained technicians with an estimated cost of about $100 per test, meaning that most people probably only get tested once. Current testing capacities are limited and results often take days or weeks to return, meaning that individuals who don’t know they are infected can transmit the virus during this time. The high sensitivity of qRT-PCR may also be a drawback rather than an advantage, because the test often detects small fragments of RNA that don’t originate from whole virus particles and thus don’t represent transmissible virus. Such RNA fragments can persist in individuals for weeks and months. As illustrated in Figure 1, infection with SARS-CoV-2 usually results in high initial levels of viral replication that peak and begin to decline within a few days. Symptoms don’t usually appear until after that peak has already occurred, and, because most people don’t get tested until they experience symptoms, they are likely already on the downward slope of viral replication and no longer infectious at the time of testing. In the meantime, they have been unknowingly transmitting the virus to others for several days. Clearly, these people need to be identified and isolated during their period of high infectivity.

In late June, Harvard epidemiologist Michael Mina published a preprint that evaluates the effectiveness of current SARS-CoV-2 surveillance measures for reducing transmission when considering frequency of testing and delayed reporting of results. Mina and co-authors concluded that infrequent testing with an ultra-sensitive test like qRT-PCR often results in unnecessary quarantine of individuals who are no longer infectious. Notably, it also results in missing pre- or asymptomatic individuals who are at the beginning of their infection and thus highly contagious, allowing them to go about their daily routines and infect others. 

A few days after publication of the preprint, Mina co-authored an opinion article in the New York Times in which he discussed the potential for controlling the SARS-CoV-2 pandemic by widespread use of frequent, rapid at-home diagnostic tests. One example of such a diagnostic test is a lateral flow device, which is a paper strip that works similarly to a pregnancy test. The strip has a sample pad on one end and contains antibodies that recognize SARS-CoV-2 antigens. One would dip the sample pad portion of the strip into a sample of saliva and allow the saliva to wick across the strip. The presence of SARS-CoV-2 antigens in the saliva would be indicated by the appearance of a test line in addition to the control line, while a negative test would only indicate the control line (Figure 2). The test provides results in 10-15 minutes at a cost of about $1-2 per test and does not require any additional equipment. A positive result would indicate the need for self-quarantine and confirmation of test results through a doctor’s office. 

Although these rapid tests are only about half as sensitive as qRT-PCR tests, they detect the presence of viral antigen during the actual window of transmissibility when viral levels are very high. The highly sensitive qRT-PCR assays detect viral RNA for weeks after a patient is no longer transmitting virus, which is irrelevant for quarantine/isolation purposes and does nothing to curb transmission. A less sensitive test that is done on a daily basis and provides immediate results would be more valuable because it would identify individuals while they are actually infectious. This would also alleviate the need for costly contact tracing measures because most infected individuals would be aware of their status and would stay isolated during their period of transmission. 

Rapid lateral flow SARS-CoV-2 diagnostic tests are already available, but there is concern that the FDA may not approve these products because of their low sensitivity. You can help bring these products to market by writing to your elected officials (see sample letter templates here), contacting your local TV and radio stations, and telling your friends and family to do the same. Hopefully, with sufficient media attention, the FDA, CDC, and NIH will recognize the value of these tests and make them widely available to the public. This may be the ultimate solution for opening schools and workplaces, and for rebuilding the economy. 

[Michael Mina discussed rapid at-home SARS-CoV-2 testing options on TWiV 640. Tidbits of that episode were also reviewed on MedCram.]