Whole parasite vaccines for the asexual blood stages of Plasmodium.

After many decades of research, an effective vaccine for malaria is still not available. Most research efforts have focused on identifying a key target antigen and then using powerful adjuvants to generate specific antibodies that can block parasites from entering host cells (hepatocytes, red blood cells). However, the inability to generate sufficiently potent antibody responses has led to significant disappointment with current vaccine programs. An additional challenge for sub-unit vaccines is that key vaccine antigens are highly polymorphic. These challenges have spurred radically different approaches to malaria vaccine development. Many of these involve the use of "whole parasites"-either extracted from mosquitoes or cultured. With these, every parasite molecule for that particular strain is included in the vaccine. This strategy is showing great promise following several clinical trials with irradiated sporozoites. However, a whole-parasite approach to a blood stage vaccine has not advanced as quickly. This article outlines the history, the different approaches that are being taken and the challenges associated with whole parasite blood stage vaccines and discusses recent exciting developments as these vaccines now move into the clinic.

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA.

Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.

Controlled Infection Immunization Using Delayed Death Drug Treatment Elicits Protective Immune Responses to Blood-Stage Malaria Parasites.

Naturally acquired immunity to malaria is robust and protective against all strains of the same species of Plasmodium This develops as a result of repeated natural infection, taking several years to develop. Evidence suggests that apoptosis of immune lymphocytes due to uncontrolled parasite growth contributes to the slow acquisition of immunity. To hasten and augment the development of natural immunity, we studied controlled infection immunization (CII) using low-dose exposure to different parasite species (Plasmodium chabaudi, P. yoelii, or P. falciparum) in two rodent systems (BALB/c and C57BL/6 mice) and in human volunteers, with drug therapy commencing at the time of initiation of infection. CIIs with infected erythrocytes and in conjunction with doxycycline or azithromycin, which are delayed death drugs targeting the parasite's apicoplast, allowed extended exposure to parasites at low levels. In turn, this induced strong protection against homologous challenge in all immunized mice. We show that P. chabaudi/P. yoelii infection initiated at the commencement of doxycycline therapy leads to cellular or antibody-mediated protective immune responses in mice, with a broad Th1 cytokine response providing the best correlate of protection against homologous and heterologous species of PlasmodiumP. falciparum CII with doxycycline was additionally tested in a pilot clinical study (n = 4) and was found to be well tolerated and immunogenic, with immunological studies primarily detecting increased cell-associated immune responses. Furthermore, we report that a single dose of the longer-acting drug, azithromycin, given to mice (n = 5) as a single subcutaneous treatment at the initiation of infection controlled P. yoelii infection and protected all mice against subsequent challenge.

Malaria vaccine design: immunological considerations.

The concept of a malaria vaccine has sparked great interest for decades; however, the challenge is proving to be a difficult one. Immune dysregulation by Plasmodium and the ability of the parasite to mutate critical epitopes in surface antigens have proved to be strong defense weapons. This has led to reconsideration of polyvalent and whole parasite strategies and ways to enhance cellular immunity to malaria that may be more likely to target conserved antigens and an expanded repertoire of antigens. These and other concepts will be discussed in this review.

Enhancing Vaccine Efficacy by Engineering a Complex Synthetic Peptide To Become a Super Immunogen.

Peptides offer enormous promise as vaccines to prevent and protect against many infectious and noninfectious diseases. However, to date, limited vaccine efficacy has been reported and none have been licensed for human use. Innovative ways to enhance their immunogenicity are being tested, but rational sequence modification as a means to improve immune responsiveness has been neglected. Our objective was to establish a two-step generic protocol to modify defined amino acids of a helical peptide epitope to create a superior immunogen. Peptide variants of p145, a conserved helical peptide epitope from the M protein of Streptococcus pyogenes, were designed by exchanging one amino acid at a time, without altering their α-helical structure, which is required for correct antigenicity. The immunogenicities of new peptides were assessed in outbred mice. Vaccine efficacy was assessed in a skin challenge and invasive disease model. Out of 86 variants of p145, seven amino acid substitutions were selected and made the basis of the design for 18 new peptides. Of these, 13 were more immunogenic than p145; 7 induced Abs with significantly higher affinity for p145 than Abs induced by p145 itself; and 1 peptide induced more than 10,000-fold greater protection following challenge than the parent peptide. This peptide also only required a single immunization (compared with three immunizations with the parent peptide) to induce complete protection against invasive streptococcal disease. This study defines a strategy to rationally improve the immunogenicity of peptides and will have broad applicability to the development of vaccines for infectious and noninfectious diseases.

Controlled Human Malaria Infection: Applications, Advances, and Challenges.

Controlled human malaria infection (CHMI) entails deliberate infection with malaria parasites either by mosquito bite or by direct injection of sporozoites or parasitized erythrocytes. When required, the resulting blood-stage infection is curtailed by the administration of antimalarial drugs. Inducing a malaria infection via inoculation with infected blood was first used as a treatment (malariotherapy) for neurosyphilis in Europe and the United States in the early 1900s. More recently, CHMI has been applied to the fields of malaria vaccine and drug development, where it is used to evaluate products in well-controlled early-phase proof-of-concept clinical studies, thus facilitating progression of only the most promising candidates for further evaluation in areas where malaria is endemic. Controlled infections have also been used to immunize against malaria infection. Historically, CHMI studies have been restricted by the need for access to insectaries housing infected mosquitoes or suitable malaria-infected individuals. Evaluation of vaccine and drug candidates has been constrained in these studies by the availability of a limited number of Plasmodium falciparum isolates. Recent advances have included cryopreservation of sporozoites, the manufacture of well-characterized and genetically distinct cultured malaria cell banks for blood-stage infection, and the availability of Plasmodium vivax-specific reagents. These advances will help to accelerate malaria vaccine and drug development by making the reagents for CHMI more widely accessible and also enabling a more rigorous evaluation with multiple parasite strains and species. Here we discuss the different applications of CHMI, recent advances in the use of CHMI, and ongoing challenges for consideration.

Vaccination with chemically attenuated Plasmodium falciparum asexual blood-stage parasites induces parasite-specific cellular immune responses in malaria-naïve volunteers: a pilot study.

BACKGROUND: The continuing morbidity and mortality associated with infection with malaria parasites highlights the urgent need for a vaccine. The efficacy of sub-unit vaccines tested in clinical trials in malaria-endemic areas has thus far been disappointing, sparking renewed interest in the whole parasite vaccine approach. We previously showed that a chemically attenuated whole parasite asexual blood-stage vaccine induced CD4+ T cell-dependent protection against challenge with homologous and heterologous parasites in rodent models of malaria. METHODS: In this current study, we evaluated the immunogenicity and safety of chemically attenuated asexual blood-stage Plasmodium falciparum (Pf) parasites in eight malaria-naïve human volunteers. Study participants received a single dose of 3 × 107 Pf pRBC that had been treated in vitro with the cyclopropylpyrolloindole analogue, tafuramycin-A. RESULTS: We demonstrate that Pf asexual blood-stage parasites that are completely attenuated are immunogenic, safe and well tolerated in malaria-naïve volunteers. Following vaccination with a single dose, species and strain transcending Plasmodium-specific T cell responses were induced in recipients. This included induction of Plasmodium-specific lymphoproliferative responses, T cells secreting the parasiticidal cytokines, IFN-γ and TNF, and CD3+CD45RO+ memory T cells. Pf-specific IgG was not detected. CONCLUSIONS: This is the first clinical study evaluating a whole parasite blood-stage malaria vaccine. Following administration of a single dose of completely attenuated Pf asexual blood-stage parasites, Plasmodium-specific T cell responses were induced while Pf-specific antibodies were not detected. These results support further evaluation of this chemically attenuated vaccine in humans. TRIAL REGISTRATION: Trial registration: ACTRN12614000228684 . Registered 4 March 2014.

Streptococcal Immunity Is Constrained by Lack of Immunological Memory following a Single Episode of Pyoderma.

The immunobiology underlying the slow acquisition of skin immunity to group A streptococci (GAS), is not understood, but attributed to specific virulence factors impeding innate immunity and significant antigenic diversity of the type-specific M-protein, hindering acquired immunity. We used a number of epidemiologically distinct GAS strains to model the development of acquired immunity. We show that infection leads to antibody responses to the serotype-specific determinants on the M-protein and profound protective immunity; however, memory B cells do not develop and immunity is rapidly lost. Furthermore, antibodies do not develop to a conserved M-protein epitope that is able to induce immunity following vaccination. However, if re-infected with the same strain within three weeks, enduring immunity and memory B-cells (MBCs) to type-specific epitopes do develop. Such MBCs can adoptively transfer protection to naïve recipients. Thus, highly protective M-protein-specific MBCs may never develop following a single episode of pyoderma, contributing to the slow acquisition of immunity and to streptococcal endemicity in at-risk populations.

The Impact of Established Immunoregulatory Networks on Vaccine Efficacy and the Development of Immunity to Malaria.

The development of vaccines to protect against parasites is difficult, in large part due to complex host-parasite interactions that have evolved over millennia. Parasitic factors such as antigenic variation and host factors such as age, transmission intensity, and genetic influences are all thought to contribute to the limited efficacy of parasite vaccines. A developing theme in field studies investigating antiparasitic immunity is the emergence, establishment, and maintenance of immunoregulatory networks that shape the immune responses to new infections, as well as vaccines, thereby influencing disease outcome. In this review, we will examine why parasite vaccine candidates perform poorly in target populations and, in particular, the role of immunoregulatory networks in influencing antimalarial immunity and vaccine efficacy. We will focus our discussion on malaria, the most important parasitic disease of humans, but also highlight the broader impact of immunoregulatory networks on vaccine efficacy.