The Future of Whole Killed Vaccines
Vaccines are the safest, most effective, and cheapest form of protection against pathogens. With COVID proving there are many different types of effective vaccines with new methods still being discovered. I wanted to take the chance to talk about one type of vaccine — whole organism killed or inactivated (WKIV). They consist of killed or inactivated cells or particles from a bacteria, virus, or parasite. These pathogens are grown in culture and then killed using heat, chemicals, or radiation to ensure they are no longer capable of replicating when placed inside a host (1).
When killed, the pathogen needs to retain its surface antigen structure or “antigenicity” so that it can maintain its stimulus with the immune system, including its ability to activate the innate, cell-mediated immune response and eventually humoral immunity. The WKIV is expected to stimulate the immune system in much the same way as the pathogen. Release of IgA and SIgA is the main goal with WKIV due to it being a first-line defense. IgA plays a mediating role with adaptive and humoral immunity, especially at mucosal sites where many pathogens enter the body (1). Priming IgA for rapid response upon reinfection is one of the most effective ways to prevent infection and colonization of pathogens (1).
There are many advantages of using WKIV. One is that they are easily transported to developing countries due to the lack of cold transport needed since they are already inactivated (2). WKIV vaccines are also considered safer due to their inability to replicate as compared to the live-attenuated vaccines (LIAV) (3).
However, there are also disadvantages as WKIV are thought to be less effective than LIAV at activating the immune response. Specifically, the cell-mediated immunity (CD4+/CD8+)and secretory IgA response are not as likely to initiate the humoral immunity required for long-term memory, which is the ultimate goal of the vaccine (2–3). These vaccines need to contain a lot more pathogen than other types of vaccines to activate the immune system and initiate the immune response, eventual protection, and memory (1,3–4). Cholera, Influenza A, and Malaria are three pathogens for which WKIV have been or are being developed due to the importance of CD4+/CD8+ T-cells, SIgA, and IgA in stopping transmission, morbidity, and/or mortality. Their efficacy and effectiveness are in constant change as new techniques are developed and they demonstrate the advantages and disadvantages of WKIV (1).
Cholera (V. Cholerae)
Vibrio cholerae is a gram-negative, highly motile bacteria with a single polar flagellum. It has one major surface antigen (O) which consists of a lipopolysaccharide. There are 206 known serotypes, with O1 and O139 being considered endemic and therefore being targeted for vaccination (5). There is a focus on WKIV and the generation of SIgA and IgA due to Cholera Toxin (CT) targeting the intestines, a major mucosal region of the body (MALT). In MALT, complement levels are low thus an unlikely mechanism of protection (6–7). Dimeric IgA is the most likely effector function in trapping Cholera, preventing colonization of the intestines, resulting in the clinical symptoms of diarrhea and vomiting that ultimately lead to mortality (7).
Protection from the orally-given WKV currently being used in Cholera-endemic areas stems from IgA production leading to a cell-mediated response, and eventual, but short-lived, humoral response (1, 7). Dukoral is a monovalent, WKV O1 V. cholerae serotype and recombinant CT B. Since it is given orally, a sodium bicarbonate buffer is also included to protect the vaccine from stomach acid (8). It also protects against E. coli (LT-ETEC) short-term due to similarities of the CT with the heat-labile protein in E. coli (5). It is known as the standard for containment and prevention from outbreaks and for travelers to endemic areas (9). Efficacy rates of the main vaccine, Dukoral, are still being debated, however, protection in children 5 years and older is better than that of children under 5, demonstrating 50–91% efficacy lasting 2–3 years (5, 10–11). Reasons for lower efficacy in young children include limited plasma cell response along with limited O-antigen response. In all age groups, TH1 and TH17 are not as prominent as they are during natural infection, and instead, TREG response is higher suggesting they may be suppressing the TH response (7, 10–12). Protection wanes off after 2 years but lasts longer in Cholera endemic areas due to a boost in memory (1, 5, 9, 11,13). Vibriocidal antibodies are used as a marker to test for protection from Cholera, as IgA testing is impractical in many settings where vaccination occurs, however, this titer is not a good indicator of protection (7). SIgA production is hard to stimulate, especially through the use of WKIV due to its short-lived nature in the body. Oftentimes, many doses are required to get the same response as a LIAV; Dukoral demonstrates this, as two doses are needed to stimulate protection (1).
There are many other WKV in development for Cholera and are described in Table 1 (5). Shanchol and Euvichol are the most well-known and studied. Shanchol provokes a more robust O antigen-specific memory B cell response which contributes to a longer duration of protection, with a similar SIgA response as Dukoral. It has been proven safe and effective for children of all ages, but protection in children 5 years and older is still much stronger. New developments in killing techniques are being tested to create a more effective antigen response, producing more IgA, leading to a more significant humoral response and longer-lasting memory (1–2). Since none of the current vaccines generate immunity amongst children younger than 5, this suggests there is a compromised response with the O-polysaccharide antigen response and therefore it has been suggested to create age-specific vaccines for improved efficacy (7).
Influenza
Influenza is a virus of the Orthomyxoviridae family, which is a single-stranded RNA (ssRNA) virus with four subtypes (A, B, C, and D). Influenza A Virus (IAV) affects humans and animals. The molecular structure involves two proteins, hemagglutinin (HA) and neuraminidase (NA) for which there are 18 HA and 11 NA subtypes known (1, 14). Due to antigenic shift, there are likely to be more novel subtypes that result in global outbreaks. Antigenic drift and shift is IAV’s primary evasion mechanism from immunity (14). Therefore, the need for an effective IAV vaccine is critical. IAV’s seasonality is critical to vaccine development and administration timing, as they are currently developed twice a year based on flu season in the Northern and Southern hemispheres (1).
Currently, there are many multivalent WIV (Influenza B, H1N1, and H3N2) used for seasonal IAV globally, whilst monovalent IAV vaccines are used to respond to particular influenza threats (i.e. H5N1) (1). The majority of IAV vaccines are replicated in embryonated hens’ eggs. The primary antigenic contents of the vaccine include various amounts of inactivated HA, NA, M, and NP protein (1, 15). Formalin or β-propiolactone are used to inactivate the virus (4). There is a range of adjuvants used depending on the country, particular age groups, and immunocompromised statuses. The vaccines are stable for one year when stored at 4–8ºC. The immune response is owed mostly to anti-HA antibodies, upregulation of CD40, CD80, and CD86 which interact with MHCII, induction of IFN-α, and inflammatory cytokines from DCs in TLR7 and MyD88 through recognition of viral ssRNA (16–18).
The antibody response is dependent on age, general health, and several other factors (18). For instance, previously exposed individuals show anti-HA IgG whereas unprimed children show systemic IgM (1). Cell-mediated immunity is also important, with CD4+ and CD8+ playing a significant role and tend to be more cross-reactive (1). Overall efficacy is around 58% depending on the season/strain, but antigenic drift brings this rate down as each season could breed a variety of genetically drifted viruses (1, 19).
There are many challenges associated with WIV including the failure to produce a significant T-cell response and SIgA response at mucosal sites, and the short-term duration of protection (14). Another challenge is the rapid antigenic drift and shift causing vaccines to be updated annually (1, 14, 18–19). Production costs and time are also significant factors in WIV, considering the inefficient propagation of virus in embryonic eggs (19).
The future of Influenza vaccines looks towards universal influenza coverage as opposed to covering individual strains (19–23). However, the variability between strains presents the biggest obstacle to achieving this goal (18). Efficacy levels of these vaccines need to be improved, as possible pandemics are looming without enough coverage to reach herd immunity (19). Future targets for a universal vaccine need to stimulate B-cells, with research showing naïve B-cells and the generation of antibodies directed against epitopes could show promise for immunocompromised individuals (15, 18). Other targets are CD8+ and CD4+ T-cells against conserved proteins to provoke viral clearance, prevent re-infection, and confer long-term immunity (15). The HA globular head has shown to be targeted for neutralization, but antigenic variation is significant, so new research points towards the HA stem to target a more conserved region. The conserved proteins that have been recognized by CD8+ include M1, M2, NP, PA, and PB1 (15). However, most of these targeting methods include vaccines that are outside the range of WIV and instead use DNA, RNA, and VLP vaccine technology (20–23).
WIV vaccines remain good candidates for a universal Influenza vaccine due to their safety and ability to activate humoral immunity while stimulating the cellular immune response (4). However, new methods of production need to be explored in order to manufacture enough vaccines in case of a future pandemic (4). Recently, a new mammalian method of production has been studied using Madin-Darby canine kidney (MDCK) for virus propagation, but so far has proved to come at a high cost with low productivity (16). More research is needed to prove new techniques effective.
Malaria (P. falciparum)
Human malaria is caused by the protozoan parasite Plasmodium. There are five species known to cause infection in humans, but only four known to be severe: P. falciparum, P. vivax, P. malariae, and P. knowlesi. The life cycle of P. falciparum is extremely complex which contributes to the severity of disease owing to its ability to infect RBCs. It also causes challenges to develop vaccines of high efficacy (1). For reference, the life cycle of malaria can be found in Appendix 1 (24).
Currently, there is only one approved vaccine against P. falciparum, RTS, S, introduced to the public in 2019. RTS, S is a subunit vaccine and is the first and only malaria vaccine. RTS, S efficacy remains low, at around 30%, with a duration of approximately 4 years, therefore research is still being conducted to find a more effective and safe alternative (25). There is currently no existing WKIV for Malaria, but this method is being reconsidered as recombinant methods are proving ineffective to combat Malaria’s genetic plasticity (26–27). Unlike other pathogens, the vaccine reproducing a similar response as the natural infection is not desirable due to Malaria’s inability to stimulate immune memory response in the short term with relatively few infections. To produce an ideal vaccine, it must provoke a stronger or different type of immune response than natural infection (27).
Challenges in Developing WKIV for P. falciparum
In the early 1900s, birds and monkeys were successfully immunized using whole Malaria parasites. This was followed by a 1970s experiment using mosquitoes to immunize humans using radiation-attenuated sporozoites, which showed some success, but was deemed impractical and the adjuvant was also not useful in humans (27–28). One of the main challenges will be finding an adjuvant that produces a similar immune response (27). A WKV is known to produce a low T-cell response which would be needed to induce robust immunity in Malaria (27). Another challenge would be producing enough parasites for the scale necessary to study and, if approved, administer to the affected areas (27). Not to mention, producing parasites is an understated ethical issue, still being heavily debated (27–28). This is especially risky when considering that a vaccine targeting one stage of the life cycle will not necessarily prevent infection or improve disease symptoms (26). As previously mentioned, the complex life-cycle, genetic variability, and plasticity are the biggest challenges to developing an effective and long-lasting vaccine (26). Yet another problem is that there is evidence showing emerging resistance to antimalarial drugs which would, simultaneously, create more urgency and complexity for vaccine development (26).
Future Possibilities
Several methods are being researched, and while many of them are recombinant, the strongest candidates for WKV include targeting the following stages in the life-cycle:
Pre-erythrocytic
Vaccines targeted at sporozoites look to generate neutralization of the sporozoite’s ability to infect and develop in hepatocytes. Both humoral and cell-mediated immunity play a role in protection from infection, disease, and transmission (1, 24, 26). Several WK sporozoite vaccines have demonstrated high levels of protection in animal models, but so far none have translated to humans (29). A trial in Mali and Burkina Faso showed low efficacy, but research is ongoing (29–30). The next likely target will be to attack the infected hepatocyte. With new antigen targets being developed, this is still a valid area of exploration for future vaccines (24, 26, 29).
Blood-Stage
Vaccines targeted at the asexual blood-stage look to use antibodies utilized in naturally acquired immunity (NAI) to neutralize merozoites before invasion or engage effector cells against antigens expressed on the schizont-infected RBC membrane (1, 24, 29). CD4 and γδ T-cells play a role in this protection naturally, and therefore should also be targeted in vaccine protection (1). Immunity would come from eliminating infected RBCs (1, 24). There has been some promise from protecting against the blood-stage using a WKV but has not shown protection from the sporozoite (28). The WK lysate vaccine showed activation of CD8+ T-cells and could contribute to the design of efficient multiple-stage subunit malaria vaccines (28). Recently, two new developments hold promise for future vaccines, including the identification of highly conserved merozoite antigens and the ability to encapsulate the attenuated-whole parasite in a MLCP-liposome and present to an APC (29, 31). More research needs to be done on targets for blood-stage immunity and effector mechanisms (26).
With blood-stage vaccines, it is necessary to culture P. falciparum in human RBCs and this is another question of ethics due to the possible induction of antibodies onto RBC membranes (31). It is also essential to provoke a much stronger immune response than NAI and with the antigens present here being prone to significant antigenic variation and plasticity mentioned earlier, the bar is high, but not unreachable (1, 24, 29).
References
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Appendix 1.
P. falciparum life cycle including the human host immune response (24).
