What reinfections mean for COVID-19
Akiko Iwasaki


One of the key questions in predicting the course of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is how well and how long the immune responses protect the host from reinfection. For some viruses, the first infection can provide lifelong immunity; for seasonal coronaviruses, protective immunity is short-lived.1
In The Lancet Infectious Diseases, Richard L Tillett and colleagues describe the first confirmed case of SARS-CoV-2 reinfection in the USA.2 A 25-year-old man from the US state of Nevada, who had no known immune disorders, had PCR-confirmed SARS-CoV-2 infection in April, 2020 (cycle threshold [Ct] value 35·24; specimen A). He recovered in quarantine, testing negative by RT-PCR at two consecutive timepoints thereafter. However, 48 days after the initial test, the patient tested positive again by RT-PCR (Ct value 35·31; specimen B). Viral genome sequencing showed that both specimens A and B belonged to clade 20C, a predominant clade seen in northern Nevada. However, the genome sequences of isolates from the first infection (specimen A) and reinfection (specimen B) differed significantly, making the chance of the virus being from the same infection small. What is worrisome is that SARS-CoV-2 reinfection resulted in worse disease than did the first infection, requiring oxygen support and hospitalisation. The patient had positive antibodies after the reinfection, but whether he had pre-existing antibody after the first infection is unknown.


This case report adds to rapidly growing evidence of COVID-19 reinfection, in which viral genomic sequences were used to confirm infections by distinct isolates of SARS-CoV-2. What do reinfection cases mean for public health and vaccination endeavors to stop the COVID-19 pandemic?
Do reinfections occur because of a scant antibody response after first infection? Of the four reinfection cases reported to date, none of the individuals had known immune deficiencies. Currently, only two individuals had serological data from the first infection and one had pre-existing antibody (IgM) against SARS-CoV-2. Because of the wide range of serological testing platforms used across the globe, it is impossible to compare results from one assay to another. For example, antibody reactivity to nucleocapsid protein indicates previous exposure to SARS-CoV-2 but not whether antibodies that can block infection (anti-spike) are present. Also, antibody levels are highly dependent on the timing after exposure. The key goal for the future is to ascertain the level and specificity of antibody to spike protein at the time of reinfection, to determine immune correlate of protection.
Does immunity protect an individual from disease on reinfection? The answer is not necessarily, because patients from Nevada and Ecuador had worse disease outcomes at reinfection than at first infection. It is important to keep in mind that the reinfection cases in general are being picked up because of symptoms and are biased towards detection of symptomatic cases. Due to the paucity of broad testing and surveillance, we do not know how frequently reinfection occurs among individuals who recovered from their first infection. Asymptomatic reinfection cases can only be picked up by routine community testing or at an airport, for example,3 and we are probably severely underestimating the number of asymptomatic reinfections. Why do some reinfections result in milder disease,3,  4 whereas others are more severe?2,  5 Further investigation is needed of pre-existing immune responses before second exposure, and viral inoculum load.
Does infection by different viral isolates mean we need a vaccine for each type? While differences in the viral genome sequence of the various isolates are a great way to know if an individual is reinfected (ruling out reactivation of lingering virus infection), it does not indicate that the second infection was due to immune evasion. There is currently no evidence that a SARS-CoV-2 variant has emerged as a result of immune evasion. For now, one vaccine will be sufficient to confer protection against all circulating variants.6 Furthermore, reinfection by a distinct viral variant from the original virus does not imply immune escape.
Does immunity prevent transmission from those who are reinfected? The Ct value of PCR correlates with viral load, and low Ct values (high viral load) might indicate infectiousness of the individual. Although Ct values can vary substantially between various tests and laboratories, in one study, samples with Ct values greater than 35 were only 8% positive for cultivable virus.7 A good proxy for infectiousness can be obtained through viral plaque assays that measure the infectious virus. However, these assays require biosafety level 3 facilities and are labour intensive, and the assays are not routinely done in clinical laboratories. Since some reinfection cases had Ct values less than 35,3,  4 infectious virus might have been harboured in the nasal cavity. Thus, reinfection cases tell us that we cannot rely on immunity acquired by natural infection to confer herd immunity; not only is this strategy lethal for many but also it is not effective. Herd immunity requires safe and effective vaccines and robust vaccination implementation.
As more cases of reinfection surface, the scientific community will have the opportunity to understand better the correlates of protection and how frequently natural infections with SARS-CoV-2 induce that level of immunity. This information is key to understanding which vaccines are capable of crossing that threshold to confer individual and herd immunity.


https://doi.org/10.1016/S1473-3099(20)30783-0

Evidence of SARS-CoV-2 re-infection with a different genotype

Dear Editor,

We read with great interest in this journal the description by Tomassini et al. of six possible cases of re-infection with SARS-CoV-2 in England.1 The characterization and extent of such re-infections are currently increasingly investigated, and their implications are a growing concern.2 Indeed, the emergence of SARS-CoV-2 in December 2019 in China was followed by the worldwide spread of the virus and its circulation for several months (https://coronavirus.jhu.edu/map.html). In several European countries, including France, the outbreak almost ended during spring, but a second COVID-19 outbreak occurred in late summer (https://covid19-country-overviews.ecdc.europa.eu/). We observed such an evolution of SARS-CoV-2 diagnoses at the Méditerranée Infection Institute in Marseille, France, where we have performed more than 300,000 SARS-CoV-2 qPCR since end of January 2020 and have detected the first infection at the end of February (https://www.mediterranee-infection.com/covid-19/). As the SARS-CoV-2 pandemic is still on-going, a current major issue is whether or not and how long immune responses to the virus are protective. In this regard, it is important to prove the cases of reinfection, which were first reported in August in Hong Kong.2 Tomassini et al. defined re-infection as qPCR positivity at least 28 days after a previous qPCR-positive Covid-19 episode that was followed by clinical recovery and at least one negative qPCR.1 We report here a patient with two infections at a 105 days interval despite seroconversion. In addition to Tomassini et al.’ criteria for re-infection, we demonstrated by genotypic analyses that the two successive infections involved distinct viral variants and that samples tested were collected from the same individual.

The patient is a 70-year-old immunocompetent man living in a retirement home due to behavioral and memory disorders. On April 22nd, 2020, he developed fever and cough. His oxygen saturation was 95%. SARS-CoV-2 PCR performed on a nasopharyngeal swab3 was positive (cycle threshold value (Ct)= 27). A low-dose chest CT-scan highlighted minimal ground glass images in both lungs. The patient subsequently fully recovered and further nasopharyngeal samples, collected on May 8th, 14th and 18th, were PCR-negative. Serological testing performed by a chemiluminescent immunoassay (CLIA) on a Liaison DiaSorin XL instrument (DiaSorin Inc., Saluggia, Italy) showed IgG seroconversion. Indeed, a serum sample collected on May 5th, two weeks after the onset of clinical symptoms and PCR diagnosis was IgG-negative, whereas a serum sample collected two weeks later, on May 18th, was IgG-positive (signal= 21; positivity threshold= 15). On August 19th, the patient was tested PCR-positive again (Ct= 18), when sampled during a systematic screening performed in his retirement home while he was asymptomatic.

Next-generation sequencing (NGS) of SARS-CoV-2 genomes was carried out using Illumina (San Diego, CA, USA) technology as previously described.3 Genome consensus sequences were generated with the CLC Genomics workbench v.7 (https://digitalinsights.qiagen.com/) by mapping NGS reads on the Wuhan-Hu-1 SARS-CoV-2 genome (GenBank accession no. NC_045512) with 0.8 and 0.9 as coverage and similarity thresholds, respectively. The genome sequence (20,879 non-contiguous nucleotides; IHU-3844/2020) from April 22nd was most closely related to those from strains of Nextrain clade 20A that circulated during the first outbreak in our geographical area4 (Fig. 1 ). The SARS-CoV-2 genome (deposited in the GISAID database (https://www.gisaid.org/) with no. France/PAC-IHU-1347/2020) from August 19th belonged to the Marseille 4 lineage that emerged in our geographical area during the second outbreak4 (Fig. 1), and 11 mutations that are hallmarks of the Marseille 4 lineage (C4543U, G5629U, G9526U, C11497U, G13993U, G15766U, A16889G, G17019U, G22992A, G28975C, G29399A) were absent from the genome obtained from the first sample. In contrast, 2 mutations (C2416U, G8371U) that are hallmarks of the genotype identified in the first sample were absent in the second genome (Supplementary Table S1). In order to prove that samples were from the same patient, we confirmed genetically that each of 24 independent short tandem repeat markers analyzed (Supplementary Material) identified identical alleles.

Fig. 1
Fig. 1
Phylogeny reconstruction based on SARS-CoV-2 genomes recovered during the first and second infections. Phylogenetic tree was reconstructed using the MEGA X software (https://www.megasoftware.net/) based on SARS-CoV-2 genome sequences, with a total of 29,703 positions in the final dataset. This analysis incorporated the genome sequences the most similar through BLASTn searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) to the two genome sequences recovered from the case-patient in April and August 2020 (indicated by a black background and a white bold font and a framed white background and a black bold font, respectively) among those obtained in our center from respiratory samples collected since end of February 2020 until end of September (indicated by a gray bold font) and those from the GISAID database (https://www.gisaid.org/) (indicated by a black bold font). Among top hit sequences from the GISAID database, a single one was kept by country. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap greater than 50% are indicated in the tree. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All nucleotide positions with less than 80% site coverage were discarded (partial deletion option). Prior nucleotide sequence alignment was performed using Muscle.

Here, we demonstrate that the same patient was infected in April, cleared the virus, seroconverted, but was re-infected four months later with a new viral variant. The two infections reflect the circulating strains in Marseille at the same time.4 It is the most comprehensive studied as it documented seroconversion following the first infection, showed drastically different viral genomes with 34 nucleotide differences, and ruled out errors of samples by techniques commonly used for forensic identifications. The present case adds to 13 previously reported cases of re-infection with a different SARS-CoV-2 strain that occurred in China, Belgium, the Netherlands, India, Ecuador and the USA2 , 5, 6, 7, 8 – 9 (Supplementary Table S2) documented with varying degrees of robustness (Supplementary Table S3). Mean age (± standard deviation) of the cases was 40±20 years (range, 24–89), and patients were mostly immunocompetent individuals (in 12 cases (86%)). The 14 reports involved men in 9 of 13 documented cases (69%). The mean delay between the two diagnoses was 81±36 days (19–142). The symptomatology of the first and second infections was much variable. In eight cases, symptoms were reported in both infections, re-infection being less severe in two cases and more severe in four, including one death. In two cases, both infections were asymptomatic, in two only re-infection was asymptomatic, and in two only re-infection was symptomatic. Serology was performed in three cases following the first infection and was positive. Serology was performed in 11 cases following the second infection and was negative in four and positive in seven.

Such early re-infections with SARS-CoV-2 is surprising, as we are used with a majority of respiratory viruses to observe a single, annual epidemic episode.10 This atypical epidemiological pattern is particularly relevant in our geographical area where the second outbreak that started during the summer was linked to multiple distinct variants having accumulated mutations that differed from viral mutants that circulated during the first outbreak.4 This deserves conducting further studies to figure out whether or not this would make sense to include several viral variants in future vaccines.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666873/

1. Coronavirus Disease 2019 (COVID-19) Re-infection by a Phylogenetically Distinct Severe Acute Respiratory Syndrome Coronavirus 2 Strain Confirmed by Whole Genome Sequencing
https://doi.org/10.1093/cid/ciaa1275

2.A Case of Early Reinfection With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
https://doi.org/10.1093/cid/ciaa1436

3. Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019

https://doi.org/10.1093/cid/ciaa1421

4. Genomic evidence for reinfection with SARS-CoV-2: a case study
https://doi.org/10.1016/S1473-3099(20)30764-7

5. Reinfection With Severe Acute Respiratory Syndrome Coronavirus 2: What Goes Around May Come Back Around
https://doi.org/10.1093/cid/ciaa1541

SARS-CoV-2 Re-infections: Lessons from Other Coronaviruses
Lia van der Hoek

Animal and human endemic coronaviruses have been known for decades, as has their capacity to re-infect. In the COVID-19 pandemic, it is key to reveal the factors that influence reinfection susceptibility. In this commentary, I provide a view on endemic animal and human coronaviruses and the correlates of protection to reinfection.
Main Text
The current rapid transmission of SARS-CoV-2 shows many signs of a so called “virgin soil” pandemic, involving a population at risk that had no previous contact with a pathogen. It is expected that patients recovered from COVID-19 will have immunity, protecting them from reinfection. This acquired immunity could, in theory, be either potent or poor. Potent immunity would indicate protection, requiring a higher dose of virus to cause an infection. Poor or no protective immunity represents a situation where waning of antibodies or immune cells results in a susceptibility similar to the one of individuals that have never been exposed to the virus. In the latter situation, reinfections may occur with every subsequent wave. In such a scenario, SARS-CoV-2 would become the fifth endemic human coronavirus, next to the four seasonal coronaviruses: HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63. Recently, the first cases of SARS-CoV-2 reinfections have been documented. In this commentary, I discuss these findings in light of our knowledge of reinfections by other human and animal coronaviruses.
Experimental Infections in Volunteers
It is generally assumed that neutralizing antibodies are protective and provide a defense to re-infection when subsequent waves cause re-exposure to the virus. However, it is important to realize that we have no actual proof that SARS-CoV-2 neutralizing antibodies (IgG and/or IgA) protect us from reinfections. Resistance to infection when experimentally re-exposed may reveal whether neutralizing antibodies indeed provide protective immunity. Such knowledge could hypothetically be found through human challenge studies; e.g., recruiting previously SARS-CoV-2-infected volunteers with neutralizing antibody titers ranging from high to low and determining by experimental infection whether high titers of neutralizing antibodies are associated with protection from reinfection. These studies have obviously not been done for SARS-CoV-2 and will probably not be done in the near future, because no rescue therapy which protects from severe COVID-19 is currently at hand. However, these kinds of studies can and have been done with the relatively harmless seasonal human coronaviruses, as these viruses only cause the common cold.
Neutralizing Antibodies and Protection to Reinfection
All studies that involved challenge with seasonal coronaviruses were done on adult volunteers, meaning they are, in fact, reinfection studies. This is because people experience their first seasonal coronavirus infection in the very first years of life, with seropositivity reaching plateau by the age of 4 to 6 years.1
In challenge studies, volunteers receive an experimental exposure to a virus via nasal drops. During the following week(s), virus shedding, increased neutralizing titers, and symptoms are documented, all seen as signs of a productive infection. These kinds of studies have been done with HCoV-229E and HCoV-OC43 from the mid-1960s to the early 1990s to study the symptoms caused by the virus or to examine immunity and therapy options. Results demonstrated that roughly half of the volunteers could not be infected by experimental exposure. Fortunately, some studies went further and examined the determinants of the observed immunity. One of the earliest studies was done with HCoV-229E in 1967.2 Bradburne et al. found that most individuals with high pre-exposure serum neutralization titers could not be infected by HCoV-229E (only 1 out of 4 persons could be infected by isolate VR-740, see Box 1 for details on virus isolates), whereas the majority of persons with low pre-exposure neutralizing titers did become infected (78%; 17 of 22). The same association between pre-existing neutralizing antibodies in serum and protection from infection was found by Callow.3 In addition, Callow also looked at pre-existing virus-specific IgAs and found that secreted IgAs in nasal washings are also associated with protection to reinfection.3 Presence of IgA on the site of entry has similarly been described for animal coronaviruses, such as porcine coronaviruses. Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) both cause severe gastroenteritis, whereas respiratory porcine respiratory coronavirus (PRCV) causes milder symptoms in the respiratory tract. An infection by any of these viruses results in production of neutralizing IgGs and local secretion of IgAs at the site of replication,4 the gut for TGEV and PEDV and the respiratory tract for PRCV. Likewise, in the case of the avian infectious bronchitis virus (IBV, a Gammacoronavirus endemic in all countries that raise chickens, for which the eye is a site of entry), virus recognizing-IgA in the lachrymal fluid (a secretion of the eye) associates with resistance against IBV reinfection in chicken.5 These studies strengthen the hypothesis of a supposed benefit of IgAs in protection against reinfections; however, IgAs are probably neither the only nor the most important factor. The closely related porcine viruses PRCV and TGEV illustrate this. An infection by PRCV induces no virus-specific IgA secretion in the gut, but does provide protection against TGEV,6 indicating that other factors such as cellular immunity and/or circulating neutralizing IgGs must provide the protection here.

Neutralizing Antibodies and Severity of Symptoms
There are two additional remarks to make about neutralizing antibodies and coronavirus diseases. It is often mentioned that people that have a reinfection, as opposed to people that have their first infection, experience milder symptoms. Although this may sound plausible, one must be aware that there are no actual data for the seasonal human coronaviruses that substantiate this. A study by Callow et al., often cited in this respect, investigated re-challenge with HCoV-229E 12 months after a first challenge with exactly the same virus7 (Box 1). The volunteers all had an asymptomatic infection, whereas their previous infection with the same isolate, 12 months earlier, showed cold-like symptoms. It must be stressed that this cannot be translated to the current SARS-CoV-2 situation, because all volunteers were adults and this was therefore not an infection into a naive person like we are now facing with SARS-CoV-2.
A second statement, said to be substantiated by the data on seasonal coronaviruses, concerns the quality of immunity raised by either a symptomatic or an asymptomatic coronavirus infection. It has been hypothesized that fewer neutralizing antibodies are produced if a coronavirus infection occurs without symptoms. It needs mentioning, however, that this hypothesis is not strengthened by data obtained from seasonal human coronaviruses. Kraaijeveld et al. and Callow et al. showed that rising neutralization titers are not dependent on the severity of symptoms. Even asymptomatic productive infections show antibody rises in the volunteers.7,8 Whether the antibody response raised by a symptom-free infection was of lower quality (e.g., lower titers or less secreted mucosal IgA) is an important question; however, it has not been examined for the seasonal coronaviruses.
T Cell Immunity
The role of T cells in vulnerability to reinfection is another important topic also not yet studied for the seasonal human coronaviruses. The very first data on virus-specific T cells recognizing seasonal coronaviruses are being generated only now, more or less as a by-product of looking at cellular immunity recognizing SARS-CoV-2. Whether CD4 or CD8 T cells play an important role in clearing human seasonal coronaviruses during the acute phase, or if immune memory B and T cells result in less disease upon reinfection, remains unknown. Cellular protection against reinfection has been investigated for one animal coronavirus. Seo et al. showed that transfer of CD8-enriched IBV-primed T cells to chicken that were subsequently IBV challenged the next day provided protection by reducing infections or, when infected, disease severity.9
Duration of Immunity to Seasonal Coronaviruses
The first human coronaviruses discovered, HCoV-229E and HCoV-OC43, were identified in the mid-1960s, and two additional seasonal coronaviruses were identified in 2004 and 2005, HCoV-NL63 and HCoV-HKU1, respectively, bringing the total to four human seasonal coronaviruses. With more than 50 years of research on seasonal coronaviruses, one would expect a wealth of knowledge on reinfections from which we can now benefit. Indeed, there are the aforementioned seasonal coronavirus challenge studies that are particularly informative, yet other early studies that looked at sero-surveillance to monitor natural reinfections are unfortunately of less use. These studies were all done prior to 2004, and because they used full virus ELISAs, which have considerable cross-reactivity for viruses within a genus, no distinction between the alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1) can be made. Therefore, only serological surveys that use species-specific serological tests, recognizing antibodies induced by one of the four seasonal coronaviruses, are informative. We very recently performed such a study in healthy adults to determine the frequency of reinfection by the same coronavirus species and found that protection to reinfection may last for one year.10 Another recent study, in healthy volunteers including both children and adults, had the unique opportunity to look at reinfection via PCR screening in respiratory samples obtained weekly. Galanti and Shaman found that reinfections by the same seasonal coronaviruses can occur in a time window shorter than 1 year.11 Regrettably, genetic information on the re-infecting strains was not obtained in either of the two studies mentioned above, and it remains therefore uncertain whether the reinfections were realized by viruses belonging to different genetic clusters of coronavirus species (see Box 2).
Box 2
Seasonal Coronaviruses versus SARS-CoV-2
Characteristics shared between seasonal coronaviruses and SARS-CoV-2
•
HCoV-OC43, HCoV-HKU1, and SARS-CoV-2 are in the same genus (Betacoronavirus)
•
Primary site of infection is the upper respiratory tract for all seasonal coronaviruses and SARS-CoV-2
•
Receptor ACE2 is used by HCoV-NL63 and SARS-CoV-2
•
Most infections are mild and do not require hospital uptake
•
One genetic type is currently circulating for SARS-CoV-2, which is also observed for HCoV-229E (at one moment in time)
Differences between seasonal coronaviruses and SARS-CoV-2
•
COVID-19 can be severe whereas diseases associated with seasonal coronaviruses are rarely life-threatening
•
The first wave of infections by SARS-CoV-2 were in naive persons, whereas seasonal coronaviruses enter primed adults
•
SARS-CoV-2 is easy to culture with fast production of progeny virus, and many SARS-CoV-2 isolates are available for research. Seasonal coronaviruses are difficult to culture in cell lines. Only three isolates of seasonal coronaviruses are as yet available for research: the Amsterdam-1 isolate of HCoV-NL63, VR-740 of HCoV-229E, and VR-1558 of HCoV-OC43
•
Thus far, SARS-CoV-2 isolates in humans belong to the same antigenic cluster. In contrast, there are two co-circulating types of HCoV-OC43, two co-circulating types of HCoV-NL63, and three co-circulating types of HCoV-HKU1, and these genetic diversities within species may represent different antigenic variants
Duration of Immunity to Animal Coronaviruses
Human coronaviruses as well as animal coronaviruses are able to re-infect their hosts. Coronavirus infections have been studied in pigs, chickens, cows, dogs, and cats, but unfortunately animal coronavirus studies have rarely monitored natural reinfections, as most of these animals tend to live a relatively short life. Studies on porcine, bovine, and avian coronaviruses, for example, investigated susceptibility to infection after vaccinations or experimental infections, yet did not investigate challenge or reinfections after a long period (>1 year). The only studies that had >1 year follow up and looked at natural reinfections are the studies done on feline coronavirus (FECV). This virus belongs to the Alphacoronavirus genus, is a close relative of TGEV, and produces mild or subclinical gastrointestinal symptoms in cats, yet can evolve into a life-threatening peritonitis. Because domestic cats live relatively long lives, reinfections could be studied. In one exceptional example in which a community was followed for more than 10 years,12 26 cats were regularly examined for rises in FECV-antibodies. The study found frequent reinfections, even up to three times in two cats. The shortest interval between subsequent infections was 11 months.12
Can Seasonal Coronaviruses Be Used as Model Systems?
The burning question is whether we can translate the abovementioned 1-year protection observed for mild endemic coronavirus reinfections to the current SARS-CoV-2 infections and development of COVID-19. There are definitely commonalities from which we may anticipate that some translations can be made, yet also some important differences (see Box 2). The first and major difference is that infections by SARS-CoV-2 can be much more severe than the seasonal coronaviruses. Proper immunological memory may be dependent on sufficient antigen exposure, and a mild COVID-19, similar to the common cold caused by the seasonal coronaviruses, may perhaps result in a 1-year protection to reinfection. In that line of thinking, persons who experienced severe COVID-19 may be protected for longer than 1 year, yet patients with mild or asymptomatic COVID-19, which comprise the majority of infections, may not. The second difference is that SARS-CoV-2 infections are new in the population, whereas seasonal coronaviruses infect previously primed adults. As mentioned above, children experience the first seasonal coronavirus infections in their first years of life. This first infection is generally mild or may even occur unnoticed, and in subsequent years repeated infections occur. We may expect that immunity to seasonal coronaviruses, due to this repeated exposure, has matured by adulthood. For SARS-CoV-2, which is now introducing itself for the first time, it remains uncertain if a single encounter is sufficient to mount good immunological memory.
Increased Susceptibility to Reinfection by Genetic Variants
In theory, if new SARS-CoV-2 strains with sufficient antigenic differences evolve, immunity may only protect against a certain antigenic variant, allowing infections with other strains. Fortunately, there is minimal antigenic diversity in the SARS-CoV-2 genome sequences today. Thus far only two mutations have reached the current consensus: the D614G mutation in the Spike and the P4715L in the ORF1ab protein. These mutations do not affect immunogenicity and all isolates co-circulating at this moment may therefore be regarded as the same type. This is like the situation for HCoV-229E. This virus, unlike the other seasonal coronaviruses, shows only chronologically distinct strains but no co-circulation of genetically different types13 (see Box 2). Considering that the HCoV-229E reinfection situation may be the situation ahead of us for SARS-CoV-2, a study by Reed, investigating HCoV-229E reinfections, becomes highly relevant. Reed found that after 8–12 months, volunteers were still immune, since there were no infections when the same isolate (see Box 1) as the one in the first challenge, was used in a re-challenge.14 Next to the homologous re-challenge, Reed also described a heterologous challenge/re-challenge experiment, 8–14 months apart using various combinations of isolates (Box 1). Cold symptoms and virus shedding were seen in 5 out of 8 volunteers upon heterologous re-challenge. In comparison with the homologous re-challenge, this shows that strain variation is influencing susceptibility to re-infections; yet, the exact combinations of virus isolates were unfortunately not provided in the manuscript. It remains therefore unknown how large the chronological distance was between strains as well as whether lab-adaptation may have influenced the results. One of the isolates used, VR-740, became lab-adapted in the 1980s, hardly causing disease,14 and may therefore not have been the best candidate virus in either challenge or re-challenge experiments. The third and final re-challenge experiment done by Reed was with an isolate, at that time suspected to be a HCoV-229E strain,14 yet in hindsight most probably HCoV-NL63.14,15 The heterologous Alphacoronavirus challenge showed a productive HCoV-229E infection in 3 of the 4 individuals previously primed with HCoV-NL6314 (see Box 1). From this it can be concluded that distinct strains of HCoV-229E, and the two distinct Alphacoronavirus species, may provide limited cross-immunity. Translating this knowledge to the COVID-19 situation reveals that we may expect little cross-protection by immunity raised by the seasonal coronaviruses. Furthermore, there will be an increased risk of reinfections when antigenically different SARS-CoV-2 strains emerge with time.
Conclusions
Endemic animal and human coronaviruses have a common characteristic: they re-infect their host. Although endemic coronaviruses have been known for decades, knowledge concerning the factors that influence susceptibility to reinfections and the severity of disease is still somewhat limited. This is in part due to the early discovery of HCoV-OC43 and HCoV-229E. At that time (mid-1960s) it was not known that half of the human seasonal coronaviruses were still unidentified. Sero-surveillance studies done before 2004/2005 (the dates of discovery for HCoV-NL63 and HCoV-HKU1) and some challenge studies with HCoV-229E are thus difficult to interpret, as HCoV-NL63 may unknowingly have interfered in HCoV-229E studies. Still, some animal and human challenge studies are highly informative, showing the importance of neutralizing antibodies (IgG and IgA) and CD8+ T cells in protection against reinfections.
Whether the current SARS-CoV-2 reinfection case reports that have been presented, some as early as a few months after the first encounter, are the rule or the exception is unknown. Data on the seasonal human coronaviruses show a protection of 1 year post infection, perhaps longer. If this is also the case for SARS-CoV-2, then we are now facing SARS-CoV-2 reinfection exceptions. However, the 1-year-or-more protection for the endemic human and animal coronaviruses may have been shaped by repeated infections from childhood on, different from what we are currently facing with SARS-CoV-2. Thus, repeated exposure may be needed to reach immunity that lasts for more than a few months. Boosting by a vaccine may then tentatively result in such an effective immunity, hopefully as active as natural exposure. Safe and effective vaccines, ideally combined with antivirals to prevent severe disease for those not immune yet, are therefore the hope we have, releasing us from lockdowns and other physical distancing policies.

https://www.cell.com/med/fulltext/S2666-6340(20)30033-7


DOI:https://doi.org/10.1016/j.medj.2020.12.005

COVID-19 reinfection: are we ready for winter?

Since the beginning of this pandemic, an ongoing concern is whether one person can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) more than once. Occasional reports of people retesting positive, after seemingly clearing the virus, have been published. In a retrospective article published in EBioMedicine in September, Jing Lu and colleagues (Guangdong Provincial Center for Disease Control and Prevention [CDC], Guangdong CDC, China), reported that 87 (14%) of the 619 patients who had recovered from laboratory-confirmed SARS-CoV-2 infection retested positive by real-time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). All patients who tested positive a second time in the study presented with mild or moderate symptoms at initial diagnosis and the researchers were unable to isolate the infectious virus by culture or detect sequencing of full-length viral genomes at the time of the second positive test. Because all patients who tested positive a second time were socially isolated during the study, the recurrence of test positivity might have resulted from ongoing shedding of viral fragments from the original infection.
Indeed, a patient who retests positive for virus might not necessarily be experiencing a second, new SARS-CoV-2 infection. To confirm reinfection with a distinct virus as opposed to prolonged viral shedding, whole viral genome sequencing from each, potentially separate, infection is necessary. Based on this standard, true reinfection cases have now been documented in Hong Kong, the Netherlands and Belgium, and Ecuador. In October, in The Lancet Infectious Diseases, Richard Tillett and colleagues (University of Nevada, NV, USA) reported the first case of reinfection in the USA. Despite the fact that isolates from both the first infection (A) and second infection (B) belonged to Clade 20C, specimen A had four single nucleotide variants (SNVs) not seen in specimen B, while specimen B had seven distinct SNVs compared with specimen A. Considering these substantial genetic differences, the authors excluded the possibility of viral evolution within the infected individual. Unlike cases in Hong Kong and the Netherlands and Belgium, the patients in Ecuador and the USA had more severe symptoms upon secondary infection. The reasons for increased severity in these two cases are as yet unknown.
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Secondary infection could result from an inadequate immune stimulation upon the first encounter with the virus, or the decline of the neutralizing antibody responses over time. In fact, the robustness and longevity of neutralizing antibody responses to SARS-CoV-2 have not yet been comprehensively defined. In the June 2020 issue of Nature Medicine, Quanxin Long and colleagues (Chongqing Medical University, China) found that, compared with the symptomatic group, patients with COVID-19 who were asymptomatic had significantly lower IgG levels in the acute phase and early convalescent phase. Moreover, 40% of patients who were asymptomatic became seronegative for IgG 8 weeks after being discharged from the hospital, compared with 12•9% who were seronegative for the symptomatic group. An insufficient immune response could be a partial reason for the reinfection case in Hong Kong, as Paul Chan and colleagues (The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China) reported in Emerging Infectious Diseases (December 2020, first online now) that the patient had low or undetectable levels of neutralizing antibody against multiple viral proteins during the primary mild-symptomatic infection and acute stage of asymptomatic reinfection.
Encouragingly, however, in a larger analysis published in Science in October, Ania Wajnberg and colleagues (Icahn School of Medicine at Mount Sinai, NY, USA), found that over 90% of infected individuals with mild-to-moderate symptoms had moderate-to-high titers of antibodies against spike protein (S-protein) of SARS-CoV-2 and these titers remain relatively stable for at least a period of around 5 months. Thus, much larger, definitive, serology studies are needed across a variety of populations and disease severities to get a comprehensive look at the degrees to which durable humoral immunity is induced by this virus.
In addition to variable immune stimulation, the evolution of SARS-CoV-2 might also have a potential role in reinfection. In a Cell paper published in September, Qianqian Li and colleagues (National Institutes for Food and Drug Control, China) systematically analyzed 80 natural variants in the S-protein for their infectivity and sensitivity to neutralization by antibody or convalescent serum samples using in vitro assays. Fortunately, the strain with the D614G mutation in the viral S-protein, a major variant now circulating globally, seems to retain susceptibility to neutralization by both convalescent serum samples and a panel of tested S-protein-specific monoclonal antibodies. This result was further supported by a medRxiv preprint done in September by Drew Weissman and colleagues (University of Pennsylvania, PA, USA). Although not formally peer reviewed, this group reported data using pseudoviruses bearing either the D614 or G614 S-protein that the D614G spike mutation could increase susceptibility of the viral S-protein to antibody neutralization. That said, Qianqian Li and colleagues found that particular S-protein variants, including A475V, L452R, V483A, and F490L, were less susceptible to neutralization by a subset of antibodies tested. Although the frequencies of such variants are still very low in the population, caution is warranted, as some leading vaccine candidates are using the S-protein as the immunogen.
In addition to humoral immunity, T cells also have a crucial role in clearing viruses from the body. In a July issue of Nature, Nina Le Bert and colleagues (Duke-NUS Medical School, Singapore) reported that CD4+ and CD8+ T cells from individuals who recovered from mild-to-severe COVID-19 could recognize multiple regions of the nucleocapsid protein (N-protein) of SARS-CoV-2. Furthermore, they found that patients who recovered from the SARS outbreak 17 years ago, which was caused by SARS-CoV, possess memory T cells that have robust cross-reactivity to the N-protein of SARS-CoV-2. Thus, stimulating T-cell responses could be an important consideration in vaccine development for a long-lasting protective effect.
With only four documented cases formally reported to date, is reinfection likely to be a rare phenomenon? Considering the relatively high percentage of asymptomatic infections (up to 40% as estimated by the USA CDC), it is possible that many undocumented reinfections have occurred. However, without viral genome sequencing data, the true reinfection rate cannot be confirmed. 12 months have passed since SARS-CoV-2 was first reported, but there is still so much to learn. Until effective methods of curing and preventing COVID-19 have been found, maintaining social distance and wearing masks are still our best options for personal protection, especially with the coming winter.
EBioMedicine

A case of SARS-CoV-2 reinfection in Ecuador
Published:November 23, 2020DOI: https://doi.org/10.1016/S1473-3099(20)30910-5

A 1 to 1000 SARS-CoV-2 reinfection proportion in members of a large healthcare provider in Israel: a preliminary report
Galit Perez, Tamar Banon, Sivan Gazit, Shay Ben Moshe, Joshua Wortsman, Daniel Grupel, Asaf Peretz, Amir Ben Tov, Gabriel Chodick, Miri Mizrahi- Reuveni, Tal Patalon
doi: https://doi.org/10.1101/2021.03.06.21253051

With more than 100 million confirmed COVID-19 cases as of March 2021, reinfection is still considered to be rare. In light of increasing reports of reinfected COVID-19 patients, the need to better understand the real risk for reinfection is critical, with potential effects on public health policies aimed at containing the spread of SARS-CoV-2. In this descriptive preliminary report, we conducted a large-scale assessment on the country level of the possible occurrence of COVID-19 reinfection within the members of a large healthcare provider in Israel. Out of 149,735 individuals with a documented positive PCR test between March 2020 and January 2021, 154 had two positive PCR tests at least 100 days apart, reflecting a reinfection proportion of 1 per 1000. Given our strict inclusion criteria, we believe these numbers represent true reinfection incidence in MHS and should be clinically regarded as such.