Leishmania for Developing a Novel Vaccine Platform: History
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Subjects: Others | Immunology
Contributor:
Kwang Poo Chang

“Bugs as drugs” in medicine encompasses the use of microbes to enhance the efficacy of vaccination, such as the delivery of vaccines by Leishmania—the protozoan etiological agent of leishmaniasis. This novel approach is appraised in light of the successful development of vaccines for Covid-19. All relevant aspects of this pandemic are summarized to provide the necessary framework in contrast to leishmaniasis.

  • Leishmania
  • Leishmaniasis
  • SARS-CoV-2
  • COVID-19
  • Singlet oxygen
  • cell inactivation
  • whole-cell vaccine platform

1. Introduction

Vaccinology, or the science of vaccines, has received a great deal of attention since the beginning of 2020. This is due completely to the prominent role it has played in response to the emergence of COVID-19—the most severe pandemic since the Spanish flu of 1918. There have been tremendous advances with unprecedented rapidity in all fields of vaccinology, including the basic science of etiological agents, vaccine designs, delivery strategies, and biotechnologies, scale-up manufacturing, clinical trials, rollout strategies, evaluation of human immune response, epidemiology database and analysis, and public health and policies.
It is an opportune time to assess the question of what we have learned from this unprecedented and overwhelming effort for future implementation in the interest of controlling infectious diseases by vaccination. This is particularly relevant to diseases, which are not as sweeping as COVID-19 but are still significant and widespread to cause localized epidemics. One example among many is leishmaniasis. This disease has been reported to occur for centuries until today, inflicting considerable morbidity and mortality on the human population worldwide, especially in resource-poor countries. One distinctive feature of leishmaniasis is the development of lasting immunity for patients after cure. The causative agents are thus useful, when rendered safe for use, not only as a whole-cell vaccine against this disease but also as a carrier with adjuvant activities or adjuvanticity for effective delivery of foreign protein vaccines against other diseases including Covid-19.

2. Current Insights

2.1. Prospect of Disease Control from the Perspective of COVID-19 Pandemic by Vaccination and Beyond

What transpired most clearly from this comparative analysis is the possibility of making multiple vaccines available for use very quickly in an emergency, like the COVID-19 pandemic. Precedence is thus set for the potential to do the same, in principle, for all infectious diseases, preferably before they turn epidemic or even pandemic. There is evidence that global warming accelerates this event for many diseases [1], including leishmaniasis with the sign of its northward expansion. Leishmaniasis is just one of the 20 different diseases targeted by WHO for elimination. While none is pandemic, together they cause significant mortality and morbidity. The incidences of these diseases are limited mainly to low- and-middle income countries, making it unlikely to garner the kind of lavish support as Operation Warp Speed. The necessity of developing vaccines against all these diseases is, however, evident [2], especially for zoonoses, like leishmaniasis or those emerging periodically from wild animals, like COVID-19. All infectious diseases with wild animal reservoirs are impossible to eradicate, requiring constant attention to mitigate their resurgence by costly preventive measures and surveillance. Vaccine development for these diseases becomes all the more urgent due to the lack of effective drugs and drug resistance.
While all events associated with anti-Covid vaccines are still unfolding, indicative of the enormity and uncertainty of such undertaking, invaluable information has been made available at a rapid pace to inform those who are interested in such a bench- to bed-side endeavor for fighting diseases. Also made evident by the pandemic-related work is the relatedness of vaccination to many facets of the disease, requiring expert input from disparate disciplines in line with the concept of the “One medicine, One health, One-world” approach. Attempts are thus made by covering all pertinent aspects of both diseases for comparison with the recognition of inevitable omissions. In the same vein, topics for further discussion are broad-based but selective to highlight and elaborate specific points of interest.

2.2. The Success of Anti-Covid Vaccines Provides Impetus to the Development of New Approaches in Vaccinology

The new approaches include Leishmania Vaccine Platform. The mRNA and adenovirus for delivering vaccines are both a novelty, although they are based on biotechnologies after refinements by extensive investigation for decades [3][4]. Mono-specific vaccines of both platforms were produced, solely containing the spike protein of SARS-CoV-2—an apparently ideal vaccine target, considering the critical role it plays in the virus entering the host cells. Indeed, vaccinated individuals were shown to produce infection-blocking anti-spike antibodies, which were found, however, to decrease in titers with time and in activities against emerging mutants. Specifically, both Delta and Omicron variants were found to replicate in and disseminate for transmission from the vaccinated. However, they were still protected from severe illness, hospitalization, and death, suggestive of the development of antibody-independent immunity. The absence of pulmonary pathology among the vaccinated is thus thought to result from the activities of vaccine-activated CD8+ T cells, which swoop in to lyse virally infected cells, thereby limiting the infection to the upper respiratory tracts. If so, Leishmania-based delivery of spike protein has the potential to strengthen such CTL activities for lasting immunity, as discussed.

2.3. The Successful Deployment of Adenoviruses as a Platform for Anti-Covid Vaccines Makes the Use of Leishmania for Vaccine Delivery all the More Conceivable

Various adenoviruses genetically engineered to cripple their replication have long been explored as vaccine carriers against a myriad of significant diseases, e.g., SARS, Ebola, Zika, influenza, AIDS, tuberculosis, and malaria [5]. The COVID-19 pandemic offered the best opportunities for clinical trials to assess various adenovirus carriers, e.g., J&J, AZ, Sputnik-5, not only for their safety but also their efficacy. Leishmania rendered non-viable by installing a double suicidal mechanism  compares favorably in safety margin versus the live albeit replication-deficient adenoviruses. While Leishmania are far more complex than viruses structurally and composition-wise as eukaryotic protozoa, the mode of their parasitism is highly specific and marked by many elements of subtleties and stealth. Indeed, leishmaniasis is thought to have evolved from a long history of host-parasite interactions for their mutual adaptations. This is suggested by the extant niche of Leishmania endoparasitism, alternating between the sand fly guts and the phagosome-lysosome vacuoles of mammalian macrophages. Leishmania exploit the endocytic mechanisms of these phagocytes for entry as their exclusive host cells, taking reclusive residence in their parasitophorous vacuoles; in contrast, pathogenic viruses, like SARS-CoV-2, break into multiple cell types and take over their biosynthetic machineries for viral replication. The adenoviral vectors are rendered non-replicative but still deliver vaccines in a similarly invasive way. Leishmania are non-toxigenic pharmacologically and elicit no acute host immune response, and so, much as certain cutaneous species, have been used as live cells for human vaccination in Leishmanization. Such immunogenic and adjuvant properties of Leishmania can now be preserved by genetic and chemical engineering to render them totally non-viable by light-inducible 1O2 inactivation. These leishmanial vectors thus possess favorable features of safety and efficacy for vaccine delivery.

2.4. The 1O2-Inactivated Leishmania are Potentially Deployable as a Platform to Deliver Multiple Vaccines from a Single or Multiple Pathogens Simultaneously Against Different Diseases

Leishmania have a large genome to accommodate foreign genes encoding desirable vaccines episomally or chromosomally and possess the eukaryotic biosynthetic machinery for their efficient transcription, translation, and post-translational modifications. Leishmania thus can express a large set of immunologically active peptide epitopes of a giving pathogen, e.g., those identified for SARS-CoV-2 [6][7]. Coupled with the natural vaccines known to exist endogenously in Leishmania, such transgenic is a de facto chimeric vaccine suitable for immunization against both COVID-19 and leishmaniasis. The 1O2-inactivation of such a chimeric vaccine is equivalent to, but better than, combining both pathogens inactivated separately by chemical or physical treatments. Inactivation of whole pathogens by these means invariably diminishes their vaccinability, as rigorous conditions are often required by necessity to ascertain safety.

2.5. Complementary to the Current Mono-Specific Anti-Covid Vaccines Is Perhaps the Use of Leishmania Transfectants Expressing Spike Protein Together with One or More Additional SARS-CoV-2 Antigens

Waning immunity and breakthrough cases of those immunized with the available monospecific vaccines are accountable, for the most part, by the mutations of the spike proteins in the emerging variants. This is well-known concerning molecules of all pathogens at the interface of host-parasite interactions. Frequent mutations of the spike protein increase the chance of their selection by immune pressures for the best fits to evade humoral immunity. Indeed, SARS-CoV-2 variants have emerged successively with an increasing number of mutations over the earlier versions in their spike proteins, e.g., up to 18 in the Delta-variant and ≥32 in the Omicron-variant. The Delta-variant became less susceptible to neutralization by previously made spike protein-specific monoclonal antibodies and by polyclonal antibodies in the sera from mRNA vaccinated cohort [8][9], accounting likely for the ease of its transmissibility or contagiousness. The Delta-variant infection was indeed reported to produce elevated viral loads by as much as 103 fold higher than those produced by the prior variants. The Omicron-variant is predicted to be even more transmissible and more vaccine-resistant, as reported from the preliminary studies by the British government scientists.
Mutations of spike proteins alone do not fully explain all the events observed, e.g., no severe clinical sequelae despite the heavy viral loads and the emergence of inflammatory immunopathology long after the infection is over. These phenomena are reminiscent of the Leishmania model for virulence [10][11], depicting separate groups of molecular determinants independently responsible for distinct phases of disease progression from infection to immunopathology to resolution. Independent up- and down-regulation of these functionally different molecules may thus produce a spectrum of clinical manifestations from asymptomatic to mild to severe cases in discordance with the pathogen loads [10].
Vaccination with Leishmania delivery of multiple antigens has the potential to foster the effectiveness in preventing the development of all signs and symptoms of COVID-19, irrespective of spike protein mutations. Of particular interest is to determine if a single dose of a multivalent vaccine is sufficient to elicit lasting immunity by this approach, mimicking the outcome of leishmanization. The emergence of mutants in successive waves seen with increasing resistance levels appears to make the vaccine formulation with spike protein alone unsustainable. Simply administering more booster shots of the same or sequence-revised vaccines is not expected to outrace the force of natural selection for the best-fit spike protein. There is, in fact, no scientific rationale against the development of multivalent vaccines from the very beginning, regardless of the platforms used.

2.6. COVID-19 Pandemic Has Brought Several Important Areas of Vaccine-Related Research into Focus for Attention

(1) Foremost perhaps is the significance of the ongoing active investigation of clinical samples for elucidating human immune response to SARS-CoV-2 infection and vaccination. Such efforts provide crucial information for real-world verification of the laboratory findings obtained at the cellular level in vitro and with animal models in vivo.
(2) The initiation of “Human challenge trials” is made possible by the urgency of the COVID-19 pandemic, akin to controlled human infection model (CHIM) already used to screen drugs/vaccines for their safety and efficacy against some infectious diseases [12]. The pandemic urgency may further incentivize technical breakthroughs to further reduce the risk of this model for regulatory compliance so that it can be used to study areas of significance directly related to human infection and immunity. One area of special interest for such investigation in CHIM is the mechanism of animal-to-human transmission of pathogens. The outcome is expected to shed light on how different pathogens jump from animals to humans relevant to the source of their origin, e.g., SARS-CoV-2. Such investigation also has the potential to explain different outcomes of pathogens’ animal-to-human transmission, e.g., highly airborne transmissibility of the virus to cause catastrophic pandemic COVID-19 versus Leishmania’s vector-transmission to cause wide-spread endemics of cutaneous leishmaniasis with limited severity. Another area of interest to study in CHIM (and in clinical trials) is the post-chemotherapy-immune response of the volunteers to the therapeutically killed pathogens in situ. Since no drug or treatment is expected to reach everywhere in the body to eliminate all targeted pathogens, immune clearance of the residual pathogens has long been considered a mandatory step for the “clinical cure” of infectious diseases after therapeutic intervention. The outcome has the potential to help design vaccines and drugs that are more effective than those in use. Pathogens assessed as weak in vaccinability may have them strengthened by using Leishmania-based delivery of their vaccine candidates.
(3) Of utmost importance are several unanswered questions relating human immunity to pathogenic infection, i.e., exactly how pathogens, like SARS-CoV-2 or Leishmania, initiate human infection to cause their respective diseases, how the vaccines designed against them elicit a human immune response, and why the longevity of immunity against re-infection varies. It has long been thought that the outcome of human immunity to infection is determined at the first moment of host-pathogen/vaccine interactions and the dynamic events after the first encounter at the cellular and molecular levels in situ. These events have defied scrutiny in the real world. Real-time imaging and time-lapsed sample collections present technical and ethical hurdles for investigation in CHIM.
The closest available model for such studies is the in vitro simulation of SARS-CoV-2 infection by lung organoid chip/microfluidic approach [13]. The novelty of this model is the design with an artificial basement membrane lining with epithelial cells outside facing the airway passage and with endothelial cells inside facing the microfluidic blood flow. The introduction of SARS-CoV-2 to the airway initiates the infection used for studying its progression and also the response to various soluble factors and immune cells loaded to the circulating fluid phase. In vitro differentiation of pluripotent stem cells provides different cell types needed for such models. Microfluidic organs-on-a-chip of 3D cell cultures are available for other tissues. They are suitable for investigating pathogens with different portals of entry, e.g., skin, liver, colon, and brain. Time-lapsed imaging is possible, and samples are collectible from such 3D models for omics studies. Meta-analysis of the omics data is within reach by computation with the assistance of AI and machine learning programs. Information from such organoid models can integrate with one another, CHIM, and patients’ clinical data to elucidate human infection and immunity.
It is feasible to apply this approach for elucidating individual variabilities to specific infections/vaccination. This is ultimately in the domain of research for personalized medicine. Acquisition of information from individualized 3D-organoid models is highly desirable. The outcome will have considerable potential to define individual differences in responses to a given infection or vaccine, thereby explaining the wide spectrum of disease clinical signs and symptoms and uncovering unforeseen rare, but severe, consequences of vaccination, e.g., VITT. The accomplishment of such feats in the future is thus expected to delineate individual disposition of a given infection, i.e., asymptomatic, mild cases, severe illness or death, as well as to vaccination, e.g., intensity and longevity of the immunity. Inching toward that aim is perhaps recent progress in completing human genome sequencing whereby >100 additional protein-coding ORFs were identified [14]. Considerable more advances are still needed for contemplating the approach mentioned toward a full understanding of individual variations in infection and immunity.
Acquisition of knowledge in human infection and immunity is indispensable to provide precise information so that scientific experts, healthcare officials, and political leaders can make the right policies rapidly and accurately for vaccination of the population and to convince the general public of its value to combat vaccine hesitancy, skepticism, and anti-vaxxerism.

This entry is adapted from the peer-reviewed paper 10.3390/vaccines10020248

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