By Haseeb Ahsan, Jong Kyung Sonn, Young Sup Lee, Salman Ul Islam, and Saifullah Khan Khalil
Coronavirus disease 2019 (COVID–19) cases, started in December 2019, caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), have spread across all the continents of the world making it a global emergency72. Infection due to SARS-COV-2 may cause a wide spectrum of symptoms, which may include mild upper respiratory tract infection and as severe as life-threatening sepsis. Microscopic examination of coronavirus reveals that it is enveloped, single-stranded RNA virus, anchored to host cell receptors via unique spikes (9 to 12 nm), giving it the appearance of solar corona. SARS-COV-2 is one of the three most notable corona viruses known for global spread of infection20,38. Bats are thought to be the natural host for SARS-CoV-2. Pangolins have also been considered a medium of infection30,64.
It has been observed in many individuals that recruited lymphocytes and monocytes combat the infection. Upon destruction of maximum viral load at site, the immune response gradually diminishes and patients recover. In other patients, immune response becomes haphazard and infection might have very serious and fatal consequences. Mortality related to COVID-19 is mainly due to acute respiratory distress syndrome (ARDS). Blood profiling of patients in intensive care unit showed that recruited and localized white blood cells generate an irregular release of multiple cytokines (interleukins [ILs]) such as IL-2, IL-6, IL-7, IL-10, tumor necrosis factor (TNF), and macrophage inflammatory protein 1α35. Cytokine storm, which is deadly uncontrolled systemic inflammatory response, contributes to the main mechanisms of ARDS18,50. It has been established that secretion of these cytokines increases myocardial injury and severity of infection71. The unusual response of body cells to cause cytokines storm and worsen the disease is shown in Figure 1.
Disease recurrence can be described as reinfection, relapse, or recrudescence. It is necessary to differentiate the term “reinfection” from other such similar words. Figure 2 shows the basic differences in the meaning and use of these terms. According to the Center for Disease Control (CDC), reinfection refers to acquisition of new infection by a person after recovery from the first infection by the same causative agent10. A yardstick for confirmation of reinfection, by CDC, requires confirmation of initial and second infections in two time periods along with supporting evidence from genome sequencing from samples of both infections9,12. All reported cases of true or confirmed reinfections present genetic differences among samples collected from one person on two occasions of primary and reinfection. Reinfections have now been reported from the Netherlands, Belgium, Ecuador, the United States, and India23,47,60,62. All these cases are confirmed reinfections verified through genetic sequencing. It is likely that more cases of reinfections will be reported as researchers are performing viral genome sequencing to differentiate prolonged viral shedding from reinfection. Pertaining to relapse, no data of genomic sequencing have been presented so far, making them unlikely to fall under the definition of reinfection. Reasons for twice positive diagnosis via polymerase chain reaction (PCR) have been attributed to remnants of nonvirulent genetic matter after infection has been cleared. A false positive test, most probably due to mixing of samples, or consistent use of disposable equipment for sample collections can also contribute to double positive test with no symptomatic presentation and other injuries observed via imaging techniques18. One study presented PCR test results of patients using ORF1ab gene as primers generate variable results upon viral mutations63. We have assembled few representative cases of COVID-19 relapse in Table 1.
Reinfection case histories with opposite outcomes
First ever case of SARS-CoV-2 reinfection was reported and documented by comparative viral genome analysis. This patient traveled to Europe for about 1 week and returned on August 15, 2020, when he was screened positive for SARS-CoV-2 at the airport inbound traveler screening program. As required by the legislation of the Hong Kong SAR, he was hospitalized for isolation. The patient had increased C-reactive protein level and immunoglobulin G (IgG) seroconversion, which suggested that the patient had acute infection. Symptomatic presentation included mild-no symptoms of disease11,45.
Another severe case of reinfection has been reported from Nevada state, USA. Patient was diagnosed with first infection in April 2020, followed by a second positive test at the end of May. The two positive tests were separated by one negative PCR test (indicates patient recovery). Nasopharyngeal swabs were analyzed for next-generation sequencing of SARS-CoV-2 genome at both instances. For confirmation that both samples were from similar sources, researcher used a short repeat tandem marker to analyze fragments. Differences in the genetic strains of the same virus suggested that patient was infected with genetically distinct strains of SARS-CoV-2 on different occasions60.
These cases question the effectiveness of immunity acquired after natural infection of COVID-19. Different outcomes of reinfection can be due to many variables, which we have enumerated in Figure 3. All the factors ranging from personal health to immune status and viral specific characteristics are responsible for varied clinical manifestations. A recently reported study has published differenced in immune responses based on gender. Their results suggest that secretion and release of proinflammatory cytokines, which are also involved in producing cytokine storm, are higher in males. Moreover, T cell-mediated response was more robust and intense in females. These results show that males are more susceptible to develop more severe form of COVID-19 infection58. Associated comorbidities are also considered as potential risk factor to acquire infection and develop severe symptoms70.
Role of Adaptive Immune System on Varied Clinical Presentation of COVID-19 Reinfection
Adaptive immunity is one of the most significant defensive lines of action against pathogens, antigens, and xenobiotics. It offers protection against subsequent exposures of the antigen causing infection on first exposure. It implies that adaptive immune system has memory of infections and their causative agents. Hence, their pivotal role in protection against repeated exposures of SARS-CoV-2 can be easily understood. It may be acquired after natural infection (measles) or deliberately by vaccination (small pox, polio). Components of adaptive immune system include humoral (antibodies) and cell-mediated immunity (T lymphocytes)2. Adaptive immune responses of other beta coronaviruses can be reviewed in relevant references34.
Activated T and B lymphocytes, which express the receptors for antigen-presenting cells (APC), mediate adaptive immune responses. One of the significant APC is dendritic cells capable of activating itself and downstream lymphocytes upon viral entry into the body13.
Rationale for the involvement of T cells in combating SARS-CoV-2 has been derived from autopsy results of affected patients that their lungs had greater deposition of T cells while the blood profiling depicted lymphopenia. These results indicate migration of lymphocytes from systemic circulation to site of infection (lungs) and also point toward their active involvement in generating immune response59. Another study wherein the researchers observed and mapped phenotypes of SARS-CoV-2 induced specific T cell responses in people with acute infections, exposed next of kin and unexposed relatives. The authors found that SARS-CoV-2 produces adequate and functionally abundant T cell responses, which are then memorized by memory T cells to counteract any future infections by the same agent. They also suggested that recurrence maybe prevented by natural infection55. Furthermore, weakened response of adaptive immune system cannot effectively halt the proinflammatory cytokines. This frailty of adaptive immune system can potentiate cytokine storm1.
T cells are responsible for cell-mediated immunity. Cytotoxic T cells are especially responsible for their direct killing action on infected cells. Helper T cells do not take part in killing cells, but their role is of prime significance in amplifying the actions of cytotoxic T cells and B cells59. Moreover, recent experimental results obtained from rodents and other nonhuman primates have shown strong evidence of natural killer (NK) cells being part of adaptive immune system. In one study, researchers have suggested that NK cells respond strongly to viral invasion and also develop a memory of the invasion. Therefore, they can be considered and explored as prospective target for vaccine development42. Importance of T cells can be imagined from recent reports that severity of COVID-19 symptoms is directly proportional to the reduction in activity and number of helper T cells, cytotoxic T cells, and NK cells. Another study reports that common feature among severely affected patients was lymphopenia inclusive of enormous decline in helper T cells49. In a recently published study on the immune responses against SARS-CoV-2, the authors found that changes in the number of central and antigen-specific response T cells occur over a time period in individuals recovered from the infection. It was interesting to note that central memory T cells reduced over a period of 6 months after infection while the number of virus-specific response T cells increased over the mentioned time span. Active release of protective cytokines from CD4+ T cells was also present over 6 months. This suggests that cell-mediated immunity persists sufficiently in recovered patients. However, the authors have displayed their concerns that the conferred immunity becomes slightly weaker as time passes by7.
The presence of comorbid conditions also affects the severity and outcome of adaptive immune responses. One recent study analyzed the relationship and interdependence of cell- and antibody-mediated immune responses in the presence and absence of other metabolic disorders such as obesity and diabetes. One clinically significant observation of the researchers was that the coordination between T cells and antibodies was greater in non-comorbid patients. Hence, the authors strengthen the previously published views of other research groups that comorbidity poses a significant risk factor for adaptive immunity against SARS-CoV-2. Production of inflammatory cytokines in these metabolic disorders (persistently high blood glucose) is thought to be responsible for weakened adaptive immunity28. Another study observed the effects of immunosuppressive agents, given in inflammatory bowel disease, on the severity of SARS-CoV-2 infection. The authors reported that these medications delayed the development of adequate immune response in three of six patients. Older age of patients was also responsible for delayed immune response. Hence, comorbid conditions and their medications affect the progress and severity of infection53.
Role of type I interferons IFNs have recently been highlighted in various studies; their significance correlates individual genetic makeup with resistance toward severity of SARS-CoV-2 infection. Significance of type I IFN has been reported in a study conducted on 659 patients with severe symptoms of SARS-CoV-2 infection. The aim of study was to link genetics with the symptomatic presentation of SARS-CoV-2 infection. Researchers correlated the symptoms severity with inborn genetic disorders in regulating type I and III IFNs. It was found that patients who have inborn antibodies against type I IFNs had severe symptoms while those with no such autoantibodies had milder forms of infection. The authors proposed that genes expressing Toll-like receptor 3 and IFN regulatory factor 7 are involved in production of type I IFNs68. Another study found that 101 tested patients with severe COVID-19 symptoms had genetic disorders that caused fluctuations in levels of type I IFNs. They found that autoimmunity against type I IFNs of all types was developed in patients with severe symptoms. The authors also suggested that SARS-CoV-2 infection activated the silent autoantibodies. Virus-induced activation of autoantibodies reduced the concentration of type I IFNs significantly that resulted in serious clinical manifestation of the disease in 101 patients. Hence, these studies might suggest that type 1 IFNs play role in developing adaptive immunity that might impart adequate protection against the reinfection too6.
T cell memory and cross-reactive immunity
An important component of adaptive immunity is memory of T lymphocytes against antigens faced on first exposure. It is one of the important mechanisms of our body that can be manipulated or enhanced to increase the defensive power against the highly contagious nature of SARS-CoV-2. A study reported that recovered patients had established memory helper T cells and memory cytotoxic T cells’ responses against SARS-CoV-2. The study also reported similar patterns of T cell memory in unexposed individuals suggesting development of cross-immunity to SARS-CoV-2 due to immune resistance to other coronaviruses22. Cross-reactive helper T cells have been detected in many unexposed individuals during many reported studies. However, the occurrence of cross-reactive immune B cells has been very rare. One study reviewed the effect of cross-reactivity of common coronaviruses and the ability of humoral immune system to develop neutralizing antibodies against SARS-CoV-2, in in vitro settings. The conclusion of their study was that cross-reactive humoral immunity from previous viral exposures is not produced40. Although cross-reactive immunity has been reported by other groups, the likelihood of its occurrence in the building of herd immunity is still a far-fetched notion32,46. A study reported their observations and analysis of 70 individuals 42 healthy, 28 patients for 5 months. Immune memory was present in previously infected individuals after 5 months. The immune memory cells were specific for spike protein. The authors also reported the presence of predominant cross-reactive CD4+ T cells in Indian population. Cross-reactive CD8+ T cells were minimal and had less conspicuous role in providing immunity. The authors believed that the presence of these cross-reactive CD4+ T cells have been responsible for lower disease severity in Indian population despite very high positive cases3.
T cell exhaustion
It is a state which explains the behavior of T lymphocytes toward the specific antigen (virus) on chronic exposure. Exhausted T cells are functionally distinct from normal activated and memory T cells. Exhausted cells exhibit poor effector function and are usually characterized by higher expression of programmed cell death protein 1 (PD-1). A retrospective study conducted in Wuhan China revealed that patients admitted in intensive care unit displayed quite high levels of exhaustion marker PD-1 in helper and cytotoxic T cells. The study also reported that patients also exhibited very high IL-10 levels indicating that IL-10 has the capacity to induce T cell exhaustion16,41. A study conducted in Italy (severely affected during first wave of COVID-19 in Europe). reported higher expressions of PD-1 and CD57 on T cells. CD57 is marker of cell senescence showing reduced cellular defensive and proliferating potential15. Although T cell exhaustion is more common in chronic infections, its importance in SARS-CoV-2 infection cannot be undermined. Studies have revealed that T cell exhaustion is the result of massive cytokine storm produced during SARS-CoV-2 infection. Similar T cells were found in cerebrospinal fluids of SARS-CoV-2-infected patients experiencing neurological symptoms. The authors reported that T cell exhaustion and less differentiated monocytic cells were responsible for weak antiviral adaptive immune responses that led to production of neurological complications26.
Neutralizing antibodies, formed against external antigens after first exposure, play pivotal role in fighting against the subsequent exposures of same causative agent. A study revealed that most of the recovered patients produced serum-neutralizing antibodies against SARS-CoV-2 surface proteins. The plasma levels of these antibodies were different in most individuals. This may be due to inter-individual differences in the physical health and immunity65. After reinfection, high avidity IgG and high titers of neutralizing antibody were found in patients’ blood. This meant that the priming of immunity from the first episode has allowed more robust antibody response during the second episode. These findings also affirm with the results of reinfection in an animal model, in which SARS-CoV-2 re-challenge resulted in a more robust neutralizing antibody response than the primary infection5,44. Also, B cell maturation after primary infection can result in long-lived plasma cells and memory B cells. During reinfection, high-affinity antibodies are produced more rapidly by the differentiation of memory B cells into plasmablasts61. Binding sites, relative affinity, and easy access to membrane antigenic proteins are all important determinants of antibody’s efficacy and spectrum. Based on these factors, design and development of therapeutic antibodies (by acquired passive immunity). will render sufficient immune response to curb the resurgence of this disease69.
Barriers to the effectiveness of neutralizing antibodies
So far, the major target for producing antibodies, against viral antigen, has been spike S protein, which is involved in anchoring and cellular entry of virus in host cell. It is the most popular candidate for production of acquired active immunity via vaccines. A research group developed S protein nanoparticles and injected them into mice to observe the immunological response. They found that high levels of neutralizing antibodies in blood plasma of mice confirming high immunogenic nature of S protein. However, the immunity offered by these antibodies was homologous, which suggests that a generalized vaccine against all or many coronaviruses is unlikely to be produced from spike protein14.
Most of the research is currently being focused on synthesis of neutralizing antibodies against the most easily accessible surface spike S protein. However, a single escape mutation in the S protein can drastically reduce the binding efficiency of antibody and render it less effective. A recently reported study showed that substitution of a single amino acid can cost adequate reduction in antibody’s antiviral activity37.
To counteract the loss of valuable financial resources and specialized professional facilities produced by escape mutations during the development of vaccine, a convergent effort toward discovery of highly immunogenic conserved sequence of viral proteins is dire need of the day. It has been reported that the amino acid sequence motif KRSFIEDLLFNKV, found in spike protein, is one of the conserved regions in coronaviridae family. The motif is partially associated with cellular entry of virus into host cell. Researchers consider this sequence as one of the most vulnerable yet conserved sequence in coronaviruses. Exploration regarding the role of this spike protein sequence is necessary to assess and confirm the degree of attachment of virus to host cell. It can be a valuable target for long-term immunity if destruction of this sequence stops viral entry into cell51.
Developing immunity solely based on production of neutralizing antibodies can offer complications. Such complications were seen during vaccine development against HIV. Most of the resistance by HIV against antibodies has been offered by the protective mechanisms of viral envelope. Extensive glycosylation followed by high steric hindrance in the conserved regions of structural proteins make it very difficult for antibodies to bind virus54. Similar mechanisms that can hurdle the antibody attachment need to be studied in SARS-CoV-2. One advantageous factor in this virus regarding vaccine development is the position and abundance of spike proteins, which are present on the envelope and not spatially hindered52. However, developing a vaccine which targets multiple epitopes including the conserved regions of virus is the most effective long-term solution. Therefore, a vaccine that can induce the production of broad-spectrum antibodies is one of the pragmatic solutions to resolve the current pandemic crisis8.
Elevated concentrations of virus specific antibodies, which are directly proportional to the severity of the disease, are suggestive of antibody-dependent immune enhancement. This peculiar behavior of antibody-mediated increase in disease severity has been observed in SARS and COVID-1966. Enhancement of immune system after infection with HIV, influenza, and SARS has been studied in tissue cultures and animals4,33,43,57. But it is best known for its influence on the immune response to the dengue virus21,24. The recent mechanisms explain that pathophysiological effect is produced via Fc-γ receptor. Viral entry and endocytosis are not mediated by angiotensin-converting enzyme 2 receptor, but by binding of virus-bound antibody to Fc-γ receptor. After internalization, virus alters cellular response by reducing Th1 response and decreasing the production of IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS). iNOS concentrations are reduced via inhibition of STAT pathways56. Alleviation of these antiviral responses is followed by viral replication in host cell that will worsen the severity of inflammation and overall disease pathology, as shown in Figure 427.
Herd immunity is indirectly acquired immunity among susceptible individuals when present in a large group of immune individuals in the same population. It is necessary for herd immunity that immunity conferred upon by natural infection not only subsides clinical manifestations but also reduce/prevent its spread. Difficulty for the production of herd immunity in COVID-19 is due to the silent spread of disease through asymptomatic patients (not quarantined). A simple mathematical study model predicts that herd immunity in a closed community will prevail when 67% of total population is immune to the virus. Although this model is based on simple and absolute assumptions (homogeneous distribution of infection followed by acquisition of sterilizing immunity in population), the results of the model are helpful because they give us an approximate target value above which the spread of infection will halt. It is pertinent to mention that development of herd immunity will play pivotal role in warding off severe forms of reinfections48. A recent study also suggests that availability of vaccine will increase the number of immune individuals, create herd immunity, and therefore reduce the rate of transmission. However, distribution of vaccine should be prioritized to the most exposed population with highest transmission rates19.
In light of the presented reports and data, we believe that we will remain vulnerable to resurgence and recurrence of this infection in the future. Inability to produce cross-reactive humoral immunity and variable strength of T cell-mediated immunity are one of the important factors, which have not been standardized yet. Also, the fact that even viral-specific antibodies might facilitate viral replication is another anticipated problem.
Pertaining to reinfections and other complications in the near future, there has been a big concern over the ability of virus to change its genomic sequence. The only and major glad tidings regarding the influence of reinfection on subsequent waves are the scarcity of its incidence. Quite a few patients have been confirmed with true reinfection. Therefore, one can safely suggest that herd immunity is being developed in majority of population. Hence, our perspective is that reinfection will have lesser effect on the severity of pandemic as it statistically represents very minute proportion of individuals. However, things should not be taken lightly because the disease may spread from immunocompetent carrier (asymptomatic) to an immunocompromised and elderly individual. Carelessness of one individual can cost the life of another individual. Verily, this pandemic has caused immense loss to families during these days of economic downfall. Therefore, until and unless the rate of development of immunocompetent individuals outpaces the rate of viral infectivity, we cannot recline and relieve ourselves from the ultimate risks of future.
References available in An Overview About the Role of Adaptive Immunity in Keeping SARS-CoV-2 Reinfections at Bay.
Viral Immunology, published by Mary Ann Liebert, Inc., delivers cutting-edge peer-reviewed research on rare, emerging, and under-studied viruses, with special focus on analyzing mutual relationships between external viruses and internal immunity. Original research, reviews, and commentaries on relevant viruses are presented in clinical, translational, and basic science articles for researchers in multiple disciplines. The above article was first published in the November 2021 issue of Viral Immunology. The views expressed here are those of the authors and are not necessarily those of Viral Immunolog, Mary Ann Liebert, Inc., publishers, or their affiliates. No endorsement of any entity or technology is implied.protein cellular