GIVEN the present extent of reinfections and breakthrough infections one is witnessing with the highly mutated and rapidly spreading Omicron variant of the COVID-19 virus, SARS-CoV-2, one might wonder what happened to the medical concept that one gained immunity through prior natural infection and/or vaccination against the virus. C oronaviruses, to which family SARS-CoV-2 belongs, are RNA viruses. RNA viruses are known to alter their genetic make-up or mutate easily as they evolve to survive and sustain themselves amidst the vast diversity of life species. They are known to infect and readily adapt to a wide range of hosts. The coronaviruses that are known to cause upper respiratory tract infections, characterised by mild illness including the common cold, have been with us for over several decades.
SARS-CoV-2 is only a little over two years into its evolution in humans and the process is ongoing, with Omicron being its latest avatar. The virus’ future course of evolution—into either not only a highly transmissible but also more lethal variant or into mild endemic forms such as the cold viruses—remains unknown. But what is unique about COVID-19 is the unprecedented speed with which vaccines have been developed, and hundreds of millions of people around the world have been inoculated even as the virus is still evolving—the world has already seen five SARS-CoV-2 variants of concern (VOCs)—and infecting people.
Both natural infection and vaccination induce varying degrees of infection-neutralising antibody responses, but the emergence of VOCs with immune-escape mutations, such as Omicron, has posed a huge medical challenge. But, at the same time, the current situation of the pandemic has presented medical researchers with the unprecedented opportunity to study the strength, durability and breadth of immune response when people are exposed to viral antigens under different circumstances. Researchers are trying to understand whether past infection with the virus or timely spaced multiple doses of vaccine or a mix of the two confers optimal antiviral immunity to SARS-CoV-2.
On January 28, the journal Nature Medicine tweeted: “Neutralising antibodies are successfully elicited against SARS-CoV-2’s VoCs, including Omicron, after three exposures to the viral spike protein, mediated by vaccination (with BNT162b2) alone or by a combination of vaccination and infection.” This tweet followed the publication of a research paper by German scientists led by Ulrike Protzer of the Institute of Virology at the Technical University of Munich. In a tweet the next day, she said: “We are proud to share our most recent data—just out in Nature Medicine . Three antigen encounters result in superior immunity—either after three vaccinations or two plus one infection event.”
By carrying out a longitudinal study of a cohort of 171 people, comprising 98 people who had contracted SARS-CoV-2 and recovered during the first wave of the pandemic in early 2020 (convalescents) and 73 people who had not been infected earlier (infection-naive), the researchers set out to discover whether the immune system “learns” over time, either through natural infection or by vaccination, to battle Omicron and other immune-escape variants with its infection-neutralising antibody responses. Both subgroups of the study were subsequently vaccinated with Pfizer-BioNTech’s mRNA-based COVID-19 vaccine BNT162b2 and were followed up for almost two years. The initial dose was given in early 2021 and the third dose in the last quarter of 2021. The first two doses were given three weeks apart (as recommended by the vaccine developers) and the third dose nine months later. Also, to compare the efficacies between once-vaccinated and twice-vaccinated people, subsets of participants were given only the first dose and the third dose.
What the research team has shown is that a total of three exposures to the spike protein—the key antigen of SARS-CoV-2—results in the production of neutralising antibodies that are not only in high quantity but also high quality in the sense that they bind to the spike protein more vigorously, resulting in a robust immune response against all VOCs, including Omicron. This applies to triple-vaccinated people, to convalescents who later had two vaccinations, and to double-vaccinated people who subsequently had a breakthrough infection either with the Delta or Omicron variant.
The genetic barrier to the viral escape from the antibody responses of the immune system is the number of antigenic determinants (epitopes) of the virus (importantly, parts the spike protein in the case of SARS-CoV-2) that the antibodies (in convalescent or in vaccinated infection-naive individuals) can target and effectively neutralise. Now, of the 50 or so mutations in the Omicron variant, 37 are on the spike protein. So it is hardly surprising that Omicron has a greater immune-evasion potential than all the earlier variants, including Delta (which led to the second wave), resulting in both reinfections and breakthrough infections.
In the study, the team essentially looked at three parameters: the concentration of antibodies (the antibody titre) to the viral spike protein, the binding strength of these antibodies, and their ability to neutralise infection of SARS-CoV-2 variants in serum samples of the two subgroups after the first, second and the third (or booster) doses of the BNT162b2 vaccine.
The latter two parameters are particularly important to estimate the extent of protective immunity. Not all types of antibodies elicited from the immune system are equally potent at providing immunity; only those that can neutralise the virus (the neutralising antibodies as they are called) provide greater protective immunity. So it is not the antibody titre but the neutralisation capacity of antibodies that is of significance. Not unexpectedly, therefore, the study found that the ability to neutralise the virus (the neutralising-antibody levels) correlated only weakly with the total antibody titre.
From this perspective, what is critical is how effectively these antibodies bind to the virus and thus disable infection. This binding strength of antibodies gets enhanced through an endogenous process called “affinity maturation”. As against the primary antibody response following an infection or vaccination (which mainly comprises IgG besides IgA and IgM antibodies), repeated exposures to the same antigen can lead to a B-cell–assisted antibody response, which produces antibodies with increased affinity to the antigen and with the ability to adapt to changes in it. This is termed affinity maturation. Over time, therefore, a secondary response can elicit antibodies with a several fold greater affinity and neutralising capacity than in a primary response. This can include the ability to neutralise emerging variants with the potential to escape primary antibodies. Estimating immune response parameters over time (following different infection events and vaccination schedules) thus assumes significance.
A total of 486 serum samples obtained at different times from the cohort subgroups were used to study the dynamics of antibodies to the spike protein and the serum neutralisation capacity against the early SARS-CoV-2 isolate called EU1 (the Spanish variant of the original Wuhan strain) and the five VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1/B.1.1.28.1), Delta (B.1.617.2) and Omicron (B.1.1.529).
The scientists employed a new high-throughput live virus assay comprising EU1 and the five VOCs isolated from COVID-19 patients. Sera from convalescents collected nine months after infection (in early 2020) showed low virus-neutralising capacity against EU1 and all the known VOCs. After the first vaccination, while convalescents showed an average 63-fold increase in virus neutralisation, titres in infection-naive individuals were indicative of little response even after the first dose. However, the neutralisation was found to increase markedly in infection-naive individuals after the second dose (given after three weeks) though this remained significantly less than the levels in twice-vaccinated convalescents.
The assay results revealed two other interesting aspects as well. In convalescents, even four and seven months after the second dose (given after a short gap of three weeks), the neutralisation capacity was similar irrespective of whether they were vaccinated once or twice. Even though the neutralisation capacity was lower in infection-naive individuals compared with convalescents, the relative abilities of the VOCs to escape neutralisation was the same in both subgroups seven months after the second dose, with the neutralisation capacity being the lowest for Omicron. This confirmed Omicron’s highest immune-escape potential amongst all the VOCs.
Remarkably, however, the study found that after the third, or booster, dose given nine months after the second dose, the virus-neutralisation capacity against all VOCs, including Omicron, jumped greatly in both infection-naive and convalescent groups. But, relatively, the neutralisation capacity was significantly higher in vaccinated convalescents (irrespective of whether vaccinated once or twice) than in vaccinated infection-naive individuals. The study thus clearly demonstrated that convalescents showed a higher virus-neutralisation capacity against all VOCs than infection-naive individuals. The study also showed that Omicron had a greater immune-escape ability than other VOCs both in convalescents and infection-naive people at all time points of the study. Oliver Keppler of the Gene Centre at Ludwig Maximilian University, Munich, and a co-author in the work, said in a statement from the university: “For Omicron, you need considerably more and better antibodies to prevent infection.”
‘Hybrid immunity’
It has been argued by many that the “hybrid immunity” conferred on convalescents who were subsequently vaccinated could provide greater protection than just infection or vaccination alone, and this study has provided ample evidence for that. An important observation of the study in this regard is the significance of an appropriate time gap between the first and the second doses. While double vaccination within a short interval of three weeks did not enhance the infection-neutralisation capacity in convalescents, the study found that a second dose after several months significantly increased the ability to counter VOCs such as Omicron.
Another important result of the study was that though the serum anti-spike IgG antibody titres reached their maximum after the first dose in convalescents and after the second dose in infection-naive individuals, the concentrations declined rapidly in both groups (more rapidly in infection-naive people) at four to seven months after vaccination even as both the groups retained substantial infection-neutralisation capacity . This showed a lack of correlation between IgG titres and infection-neutralisation capacity.
To understand what determines the levels of neutralising antibodies, the team analysed the neutralisation activity in the study subjects against all the VOCs over time. For one, it was found that that the neutralisation capacity of both the groups against all VOCs, which was particularly low against Omicron in infection-naive individuals, increased significantly after the third dose. “Collectively,” the scientists say in their paper, “these results suggest … a maturation of antibody responses over time and after each encounter with the SARS-CoV-2 spike protein.”
Further quantitative analysis of the antibody binding strength to the spike-protein showed that in convalescents the strength increased after the first dose and remained stable thereafter and did not increase even after the booster shot, implying a maturation or maximal binding strength of spike-specific antibodies had been reached after the first vaccination dose itself. In infection-naive individuals, however, the binding affinity increased only after the second dose, and maturation, or maximal binding, was achieved only after the third dose. On the basis of these results, the authors posit that an increase in antibody binding strength to the spike protein is critical for highly effective infection-neutralisation. From this perspective, the results show the exceptional benefit of the booster shot for infection-naive individuals or two shots in convalescents (with the appropriate time gap) to counteract VOCs with high immune-escape potential such as Omicron.
The key finding of the longitudinal cohort study is that three separate exposures to the antigen spike protein—either from three doses of vaccine alone or from infection followed by two doses—conferred high immune protection against SARS-CoV-2, including its VOCs. To find out whether this result had implications for a real-world setting where the three exposures could arise from two doses of the vaccine and a breakthrough infection either with Delta or Omicron, the scientists investigated another cohort, this time comprising 31 twice-vaccinated people of whom 16 had had a breakthrough infection with Delta and 15 with Omicron.
In this second cohort, the team measured the infection-neutralisation titres after about seven days following a PCR-based diagnosis of a breakthrough infection. It was found that the neutralisation titres were significantly higher among these 31 individuals than among twice-vaccinated infection-naive participants in the first cohort. The levels were comparable to those found in twice-vaccinated convalescents or thrice-vaccinated infection-naive individuals of the first cohort two weeks after the last dose. The study did not find any significant difference in the infection-neutralisation capacity against the various VOCs, including Omicron, between those with Delta and Omicron breakthrough infections. It was also found, though with not as much statistical significance, that following breakthrough infections there was increasing antibody binding strength to the spike antigen over time.
The two cohort studies taken together thus imply that a total of three timely spaced exposures of the immune system to the SARS-CoV-2 spike protein (of the Wuhan or wild type strain or mutated variants), irrespective of the type of exposure, leads to superior infection-neutralisation capacity. “In all cases, the neutralisation activity reached similarly high levels and this was paralleled by an increased binding strength of the antibodies,” said Keppler in the statement.
“The immunity built up or strengthened by means of vaccination is key to effective protection against future variants of the virus,” said Ulrike Protzer. “A recent breakthrough infection has, in fact, the same effect as an additional vaccination on this important [humoral] arm of the immune system,” she said, commenting on how “hybrid immunity” could be achieved. “The more rapid induction of high-avidity [binding strength] antibodies in convalescents after vaccination can be compensated for by three vaccinations in infection-naive individuals, and [which] also develops after a breakthrough infection in twice-vaccinated individuals,” the research paper notes.
Washington study
The finding that breakthrough infections spur strong antibody responses was also reported in the journal Cell by a research group led by Alexander Walls and David Veesler from the University of Washington School of Medicine. The Washington study too found that those who had completed a three-vaccination protocol, convalescents who had been vaccinated after recovery and those with a breakthrough infection after vaccination launched almost comparable neutralising antibody responses in terms of magnitude and breadth.
This study too showed that serum-binding and antibody-neutralising responses to different VOCs were much more potent and lasting than those generated by people who had received only two doses of a COVID-19 vaccine or who had had a previous infection not followed by vaccination. The team investigated neutralisation of the Omicron variant in particular and found a high degree of immune evasion, but the booster dose helped close the neutralising-antibody gap caused by Omicron (see figure). While the Munich group had limited its study to one vaccine, the Washington team studied vaccines based on different platforms, including Covishield/AstraZeneca. However, the study was limited by its sample size, which was only 15. The Washington study, however, significantly found that repeated exposure to the SARS-CoV-2 spike protein did not improve the immune response to more divergent coronaviruses like SARS-CoV-1 or the different common cold viruses such as OC43 and HKU1.
While the Munich study did broaden its analysis of the humoral immune response to infection by including in it the memory B-cell–assisted antibody response and the role of affinity maturation over time, it did not look at the cellular arm of the immune system. The analysis did not include studying the role of T-cells in mounting an immune response over time, which scientists have argued would play an important role in preventing serious COVID-19 disease.
Memory T-cells detect and destroy virus-infected cells after antibodies from the primary immune response have waned with time. The so-called “helper T-cells” also prompt B-cells to produce fresh loads of neutralising antibodies. “Although the development of infection-neutralisation capacity mediated by spike-specific antibodies and antiviral T-cell immunity has been shown to develop in parallel, further studies are required to elucidate whether three timely spaced encounters with spike antigen also go along with a quantitative and qualitative increase in protective T-cell immunity,” the Munich authors note in their paper.
Nimesh Gupta of the National Institute of Immunology, New Delhi, was quoted in the daily The Telegraph (India) as saying that the institute was already pursuing such a study. “It is likely that three doses or a combination of infection and two doses will also strengthen the magnitude and quality of memory T-cell responses,” he has been quoted as saying.
From an Indian perspective, what lessons do the above two studies give? While at a general level, these do provide pointers for the national COVID-19 vaccine policy, it is important to have more specific data for Indian conditions, especially given that the vaccines available in India are different and that the seropositivity in the general population is high, nearly 70 per cent according to the National Serosurvey data of July 2021. Therefore, following widespread vaccination with well-spaced doses, most Indians would have encountered the spike protein thrice already and are likely to have acquired a fair level of hybrid immunity. So a booster vaccination policy would do well to take this aspect into account. A longitudinal study of the Munich kind would have served to evolve a science-based vaccine policy. Unfortunately, there have been no such studies so far, nor any that seem to be ongoing or being planned despite the high incidence of breakthrough infections with Delta and Omicron.
The only study on breakthrough infections in the country seems to be the one led by scientists from the National Institute of Virology, Pune, of the Indian Council of Medical Research on 39 cases of breakthrough infections with Omicron, which involved 28 travellers from abroad (and who had received vaccine doses available abroad) and their 11 high-risk contacts. This work was published in the online preprint archive bioRXiv on January 25 and is yet to be published in any peer-reviewed journal. The studied group comprised 25 individuals doubly vaccinated with the AstraZeneca vaccine (not Covishield), 8 who were vaccinated with the Pfizer-BioNTech vaccine BNT162b2 and 6 who were unvaccinated.
Using the sera of the group following the Omicron breakthrough infection, the study analysed the IgG and neutralising-antibody responses against the key epitopes of all VOCs. The researchers found that there was a threefold reduction in the neutralising antibody levels in breakthrough individuals vaccinated with the BNT162b2 vaccine as compared to those vaccinated with the AstraZeneca vaccine. But the more important result from this study is that individuals infected with Omicron had a significant immune response that could neutralise not only the Omicron variant but also the other VOCs, including Delta. This implies that the immune response induced by Omicron would make reinfection with Delta unlikely.
The authors have, therefore, emphasised the need for an Omicron-specific vaccine strategy. While the result is interesting, it is limited by its small sample size. Moreover, this study was based on foreign vaccines and subjects from abroad. How applicable their findings can be to the Indian context needs further evaluation through studies with Indian nationals vaccinated with vaccines available in the country.
