Human onchocerciasis or river blindness is a serious parasitic disease caused by infection of the filarial nematode Onchocerca volvulus transmitted through the bite of an infected blackfly (Lustigman, 2018). It is a devastating neglected tropical disease with an adverse impact on human health and socioeconomic status because it causes irreversible visual impairment or blindness and stigmatizing dermatitis (Schwartz et al., 2020). Some evidence indicates that onchocerciasis may also cause seizures and epilepsy (Romo, 2020). More than 32 million people were estimated to be infected with O. volvulus, most of whom live in sub-Saharan Africa, before the mass drug administration (MDA) with ivermectin was initiated in 1995 (Coffeng et al., 2013). Approximately 385,000 suffered from blindness, 944,000 from low vision, and 3 million from severe itching (WHO, 2010). Even though the implementation of MDA with ivermectin and vector control results in a significant reduction in the global prevalence of onchocerciasis, it remains highly prevalent. Recent estimates from the Global Burden of Disease Study 2019 indicate that 19.1 million people live with onchocerciasis with the highest prevalence and disease burdens in the Democratic Republic of the Congo and neighboring Central African Republic, South Sudan, Chad, and Cameroon (Institute for Health Metrics and Evaluation). In these endemic areas on the African continent, onchocerciasis results in an estimated 1.23 million years lost from disability due to debilitating effects of skin disease and vision impairment (G. B. D. Disease Injury Incidence and Prevalence Collaborators, 2018). In 2019, more than 200 million people in 30 countries required preventive therapy for onchocerciasis (WHO, 2020).

MDA using ivermectin alone may not be sufficient to achieve onchocerciasis elimination due to several key barriers. First, MDA with ivermectin cannot be implemented in areas co-endemic with onchocerciasis and loiasis due to the risk of severe adverse events (SAEs) including encephalopathy in patients infected with filarial Loa loa treated with ivermectin (Gardon et al., 1997; Kelly-Hope et al., 2014). Failure to control onchocerciasis in these regions allows the establishment of reservoirs of Onchocerca infection to spread into neighboring areas that previously received MDA (Kelly-Hope et al., 2014). Another concern for ivermectin MDA is the emergence and/or potential widespread of partial or complete ivermectin drug-resistant O. volvulus, which may reduce the long-term effectiveness of ivermectin when used alone in the endemic regions (Lustigman and McCarter, 2007; Turner et al., 2013). Based on these limitations, a disease modeling study concluded that it may not be possible to eliminate onchocerciasis even after 50 years of annual ivermectin MDA; this has prompted suggestions to adopt a biannual treatment program to reach the goal of the London Declaration on Neglected Tropical Diseases Goals for onchocerciasis (Dadzie et al., 2003; Turner et al., 2014).

Based on these limitations, the development of new tools such as new drugs and vaccines will be necessary to ensure sustainable onchocerciasis elimination (Hotez et al., 2015; Lustigman, 2018), as proposed in the WHO, 2021–2030 Roadmap on Neglected Tropical Diseases (WHO, 2021). The development of prophylactic and therapeutic vaccines will complement current and future chemotherapeutic programs and thus accelerate the elimination efforts of onchocerciasis worldwide (Hotez et al., 2015; Makepeace et al., 2015). The efforts to develop prophylactic and/or therapeutic vaccines for onchocerciasis have been explored since the late 1980s by many researchers and entities, and here we summarize the recent advances made in the development of vaccines against the infection of O. volvulus and onchocerciasis.

A major obstacle for vaccine development against O. volvulus infection was the lack of a suitable animal model used to evaluate the immunogenicity and vaccine efficacy of human vaccines. The O. volvulus parasite only naturally infects human and gorillas and has rarely been observed in other primates (Vandenberghe et al., 1964; Duke, 1980; Abraham et al., 2002). Even though some primates were able to be experimentally infected with O. volvulus and in which adult filarial worms can develop (Eberhard et al., 1991; Eberhard et al., 1995), it is not feasible or practical to use non-human primates as an animal model on which to test a vaccine against onchocerciasis at the early stage due to high cost, logistical limitations, and ethical constraints (Abraham et al., 2002; Lustigman et al., 2002; Abraham et al., 2021). Another limitation for vaccine development against onchocerciasis is the difficulty to obtain the third-stage infective larvae (L3) without definitive animal hosts infected with the parasite to provide the microfilariae needed to infect blackflies. Infected blackflies by feeding on consented infected individuals are the only source of infective L3. The success in the cryopreservation of infective larvae of O. volvulus greatly facilitated the ability to conduct challenge studies in the laboratory (Trpis et al., 1993) using the following models.

There is strong epidemiological evidence showing that acquired immunity against O. volvulus infection occurs in some people living in endemic areas. The population in hyperendemic regions with age >40 had a trend of decreased skin microfilariae (mf) density (Duke and Moore, 1968) or reduced number of adult worm-caused skin nodules (Albiez et al., 1984), indicating that chronic infections with O. volvulus may have developed a means of limiting further infections, although some of the individuals could be less susceptible to worm infection irrelevant to adaptive immunity. About 1%–5% of the population in hyperendemic areas do not exhibit patent infection or any clinical symptoms after living in endemic areas for extended periods of time and keeping constant exposure to the infection (Gallin et al., 1988; MacDonald et al., 2002). These individuals are believed to acquire immunity against infection of O. volvulus referred to as putatively immune (PI) (Elson et al., 1994). Another indication of acquired immunity is concomitant immunity, induced by the current infection to prevent newly infective L3 from developing into adult worms in order to keep a stable adult worm burden (Mitchell, 1990; MacDonald et al., 2002). This immunity is directed not against the concurrently infected adult worms or the microfilaria in infected subjects but against the reinfection with new L3. The acquired immunity was also observed in cattle. The cattle raised in endemic areas without detectable parasites were significantly less susceptible to the challenge with O. ochengi than age-matched naïve cattle (Tchakoute et al., 2006).

Understanding the immunological mechanisms underlying the protective immunity in PI subjects can lead to the identification of antigens involved in the protective immunity as vaccine targets against this infection. Immune sera collected from PI people specifically recognized many antigens in L3, L4, or microfilaria compared to sera from infected people, indicating that PI develop unique antibody responses that may be related to the protective immunity (Irvine et al., 1997). Previous studies have shown some associations between immunity and defined antibody and/or cellular responses in exposed individuals. Infection with O. volvulus induced strong IgG and subtype IgG responses to a 20-kDa antigen (GP20) in O. volvulus, especially with a high level of IgG3 in putatively immune persons, which indicates its importance in acquired immunity to onchocerciasis (Boyer et al., 1991; Stewart et al., 1995). Cellular response measurement demonstrated that PBMCs collected from PI individuals produced enhanced levels of IL-5 (Th-2), INF-ɣ (Th-1), and a higher level of GM-CSF responses than the infected group against O. volvulus larval/adult antigens, indicating that those PI have distinct antigen-specific cytokine responses (Turaga et al., 2000). PI individuals showed significantly higher antigen-specific IL-2 (Ward et al., 1988) and INF-Υ (Elson et al., 1995), indicating that Th-1 like responses are associated with immunity to onchocerciasis in putatively immune persons; however, other researchers did not find the correlation of cellular response with protective immunity in PBMCs from PI subjects (Gallin et al., 1988). All results indicate that putative immunity is associated with mixed Th1/Th2 responses against O. volvulus larvae (Greene et al., 1985; Turaga et al., 2000; MacDonald et al., 2002).

Another study observed that neutrophils or other immune effective cells (e.g., eosinophils) collected from endemic-infected or non-infected individuals (PI) adhered to infective L3 in the presence of specific IgG in their sera; it promoted the killing of L3 or inhibited the molting of L3 to fourth-stage larvae in vitro ( Figure 1 ), indicating that this antibody-dependent cell-mediated cytotoxicity (ADCC) against L3 is involved in the immunological defense mechanism (Leke et al., 1989; Johnson et al., 1991; Johnson et al., 1994). These findings further suggest that specific protective immunity may occur in O. volvulus natural infection.

Infective larval worms that undergo attenuation through radiation, ultraviolet light, or other means have been used experimentally to investigate their vaccine efficacy and commercially to produce veterinary vaccines against canine ancylostomiasis (hookworm infection) (Steves et al., 1973) and bovine dictyocaulus lungworm infection (McKeand, 2000). UV-irradiated Ascaris suum eggs containing the infective larvae also induced significant protective immunity against a challenge infection (Urban and Tromba, 1984). They provide an early indication for the feasibility of larval antigens to induce protective immunity for human helminth infections, and this strategy was adopted as an initial approach for a human hookworm vaccine initiative (Schneider et al., 2011).

This approach was also used to determine if protective immunity can be induced by the irradiated L3 of O. volvulus at the early vaccine development stage. Mice immunized with a single dose of irradiated L3 of O. volvulus significantly reduced the survival of challenged O. volvulus L3 in implanted diffusion chambers; however, killed L3 could not induce the protective immunity indicating that irradiated L3 induces protective immunity that requires L3 to be alive, possibly related to the fact that living L3 can secrete proteins (Lange et al., 1993). Immunization induced by irradiated L3 induced high levels of antibody responses, including IgG1, IgG2a/2b, IgA, IgE, and IgM, which recognized O. volvulus L3 and adult antigens (Yutanawiboonchai et al., 1996). The killing of challenged larvae in immunized mice required direct contact with host immune cells, especially eosinophils. The protective immunity was associated with increased levels of IL-5, IL-4 (Lange et al., 1994), and IgE (Abraham et al., 2004), but not with INF-Υ response (Lange et al., 1994; Johnson et al., 1998), indicating that Th-2 response and eosinophil effector cells are important for the irradiated L3-induced protective immunity against a challenge infection with O. volvulus.

Cattle immunized with irradiated L3 of O. ochengi acquired sterile protection against experimental challenge and significant protection against natural infection in the field with a significantly reduced nodule load and microfilaria count as well. These results provide evidence of protective immunity induced by the irradiated L3 and the proof of principle for immunoprophylaxis under experimental challenge or field natural infection (Tchakoute et al., 2006). The proof of principle for vaccine efficacy against onchocerciasis in naturally exposed cattle has been also demonstrated against O. lienalis in cattle vaccinated with microfilaria extracts (Townson and Bianco, 1982).

Although significant or complete protection can be induced by the immunization of irradiated L3 against O. volvulus infection, the safety concern for the use of living larvae and the difficulties to obtain large quantity of infective L3 from infected blackflies has limited its application in human vaccine. However, the immune sera from animals immunized with irradiated L3 have been used to identify those L3 antigens potentially involved in the protective immunity and thus promising vaccine candidates. A similar approach was used to identify the first human hookworm vaccine candidate to enter phase 1 clinical trials, an antigen known as Na-ASP-2 (Bethony et al., 2008), before revising human hookworm vaccine candidates to focus on adult stage antigens (Adegnika et al., 2021).

Both prophylactic and therapeutic vaccines have been considered for control of onchocerciasis. An effective prophylactic vaccine should protect vulnerable populations living in endemic areas against O. volvulus new infections, especially for children 1–5 years old before being exposed to the infection. The therapeutic vaccine aims at reducing the adult worm burden and microfilariadermia that cause pathology. Both preventive and therapeutic vaccines could also contribute to lower transmission rates in the endemic region to complement and enhance the MDA with ivermectin endgame or reduce the use of ivermectin, thus potentially reducing the emergence of drug resistance (Lustigman et al., 2017).

A traditional roadmap to develop a vaccine against infectious diseases includes identifying vaccine candidates through immunological approaches to identify antigens recognized by the protective immune sera. Immunoscreening of a stage-specific cDNA library of O. volvulus with the sera from individuals who acquired protective immunity against infection after many years of exposure to natural infection in endemic areas or developed concomitant immunity (Lustigman et al., 2002) is the major approach to identifying the immunodominant antigens with potential as vaccine candidates. The antigens recognized by these protective sera could be the promising targets of prophylactic and/or therapeutic vaccines against O. volvulus infection (Abraham et al., 2021). The construction of cDNA libraries from critical life-cycle stages for the establishment of O. volvulus infection including infective L3 and the molting L3 (mL3), or microfilariae (Mf), has provided an important platform to screen for such vaccine targets.

However, some protective antigens with essential functions to maintain the life cycle of parasites may not be exposed to the host immune system during natural infection, such as digestive proteases that coat on the intestinal tract of the target parasite. These “hidden antigens” with essential functions cannot be identified using conventional immunological approaches (Hotez et al., 2016). In addition, parasitic helminths secrete a lot of proteins with immunomodulatory functions to reduce host immune response as a survival strategy to evade host immune attack. These immunomodulatory proteins may not induce a strong immune response during natural infection and therefore may not be identified by the immunological approach (McSorley et al., 2013). Biochemical or functional analyses of parasite molecules using newly developed proteomics, secretomics, and metabolomics technologies provide new approaches to identify vaccine candidates with functions essential for the survival of parasites. Many functional proteins have been identified in the excretory/secretory products (ESPs) of O. volvulus that have potential as vaccine candidates (Vanhamme et al., 2020).

As more genome and transcriptome databases for Onchocerca spp. and other related nematodes become available, a faster and novel approach to vaccine discovery from genome analysis known as “reverse vaccinology” has been adopted to design vaccines against parasites (Kanampalliwar, 2020). By using bioinformatics tools, the whole genome sequences of a pathogen or related pathogens can be in silico analyzed and screened to identify DNA sequences that encode proteins bearing essential functions and vaccine properties, or immunogenic epitopes for making multi-epitope vaccines. The enormous genome sequencing information and the rise of bioinformatics have triggered a new era of vaccine research and development of the so-called “third generation” of vaccinology (de la Fuente and Contreras, 2021). Transcriptomic analyses of L3, mL3, Mf, and adult filarial stages also accelerate the identification of stage-specific antigens (Bennuru et al., 2016) and, accordingly, could identify genes essential for the early establishment of infection in the host, therefore the important targets for prophylactic vaccines of onchocerciasis (Lizotte-Waniewski et al., 2000). The overall roadmap to making vaccines against onchocerciasis is summarized in Figure 2 .

Based on the observations highlighted above, two strategies are extensively used to identify antigens from O. volvulus with vaccine potential. One is the immunoscreening of stage-specific cDNA libraries including those critical for establishment of O. volvulus infection (L3 and mL3) with sera from people living in endemic areas who are putatively immune or from animals immunized with irradiated L3 and acquired protective immunity against challenge infection (Lustigman et al., 2002; Abraham et al., 2021). Another is to identify functional proteins that are essential during the infection process or maintenance of the life cycle in the host by analyzing the O. volvulus genome or transcriptome (Lizotte-Waniewski et al., 2000) or by studying functional homologs that induce protective immunity in other nematode host–parasite systems (Gregory et al., 2000). Some of the functional proteins were found to be present as excretory/secretory products (ESPs) in the nodule fluid or secreted by L3 during infections. Analysis of ESPs in nodule fluid in cows infected with O. ochengi identified 85 proteins with drug or vaccine potential (Eberle et al., 2015). Vaccination with these crucial molecules may induce immune responses that block or interfere with the establishment of the parasite in the host (Lustigman et al., 2017; Abraham et al., 2021). Another 26 protein antigens were identified through immunoscreening of a cDNA library with different protective sera, 12 of which demonstrated partial or significant protection when tested in the O. volvulus mouse model (Abraham et al., 2001; Lustigman et al., 2002; Lustigman et al., 2003). Following are the major identified vaccine candidates for onchocerciasis ( Table 1 ).

To down-select the antigen(s) with the most promising vaccine efficacy as vaccine candidate(s), the following criteria were used to create a priority ranking: (i) with known or essential function required in the infection and survival in the host; (ii) L3 or L4 abundant antigens responsible for the initiation of infection in host; (iii) localized on the surface area immunologically accessible to the immune responses; (iv) recognized by the antibodies from humans with putatively protective immunity (PI) or cattle, chimpanzees, and mice immunized with irradiated larvae; (v) protection identified in a mouse chamber model or the killing (or L3 molting inhibition) of larvae by the antibody in the presence of neutrophil or monocytes in vitro; and (vi) presence of the protective homologues in other filarial or nematodes (Lustigman et al., 2002; Lustigman et al., 2017).

Based on the selected criteria, 8 of the 12 potential vaccine candidates described above that induced worm reduction between 39% and 69% (Abraham et al., 2001; Lustigman et al., 2002; Lustigman et al., 2003; Lustigman et al., 2017) were selected for further evaluation in the mouse chamber model in parallel under the same conditions. These 8 antigens are Ov-CPI-2, Ov-ASP-1, Ov-RAL-2, Ov-ALT-1, Ov-103, Ov-B20, Ov-FBP-1, and Ov-CHI (Lustigman et al., 2017). To avoid the potential side effect of the functional cysteine protease inhibitor of Ov-CPI-2 (Gregory and Maizels, 2008) and to increase its immunogenicity in immunized hosts (Babayan et al., 2012), the Asn66 responsible for the asparaginyl endopeptidase inhibitory activity was mutated to Lys66 (Babayan et al., 2012) and thus created a mutated form of the inhibitor, Ov-CPI-1M. Seven of the eight down-selected antigens were expressed as soluble recombinant proteins in yeast Pichia pastoris and E. coli systems at Texas Children’s Hospital Center for Vaccine Development under strict conditions of quality control. Mice were immunized subcutaneously with each of the purified recombinant proteins formulated with Rehydragel™ LV (alum) for three times before being challenged with L3 in the embedded diffusion chamber. Out of seven vaccine candidates tested, Ov-103, Ov-RAL-2, and Ov-CPI-2M induced protective immunity against O. volvulus L3 and the protection was reproducible in separate trials (Hess et al., 2014). Mice immunized with recombinant Ov-103 expressed in yeast induced a significant 30% reduction in L3 survival and a 63% level of host protection (mice with parasite recovery lower than control mice) while recombinant Ov-103 expressed in E. coli did not show significant protection. Interestingly, mice immunized with E. coli expressed recombinant Ov-RAL-2 induced a significantly higher protection than protein expressed in yeast (L3 survival reduction 39% vs. 24%, host protection 64% vs. 55%). These protection differences between proteins expressed in E. coli and yeast for both Ov-103 and Ov-RAL-2 seem not to relate to the serological IgG titers and molecular structures because both Ov-103 and Ov-RAL-2 expressed in E. coli and yeast displayed similar and comparable α-helix structures and induced similar levels of IgG (Hess et al., 2014). However, mice immunized with Ov-CPI-2M expressed in both E. coli and yeast produced similar protection against O. volvulus L3 challenge in the diffusion chamber (30% L3 survival reduction and 17% host protection) with similar levels of antigen-specific IgG levels (Hess et al., 2014). To test for synergy of efficacy when the antigens are combined, fusion of Ov-103 and Ov-RAL-2 or Ov-103/Ov-RAL-2/Ov-CPI-2M was produced. The challenge test with the fusion revealed similar levels of protection induced by each individual protein, and there was no increased protective immunity observed in the antigen combination (Hess et al., 2014).

Confirmatory protection with adjuvanted Bm-103 and Bm-RAL-2, the B. malayi homologues of Ov-103 and Ov-RAL-2, was determined using the gerbil model after challenge with B. malayi L3 larvae subcutaneously. Gerbils immunized with yeast-expressed recombinant Bm-103 or E. coli-expressed recombinant Bm-RAL-2 produced 39% and 42% early worm reduction 42 days post challenge with B. malayi L3 and 22% or 46% reduction of adult worm burden (150 days), respectively. Vaccination with a fusion Bm-103 + Bm-RAL-2 expressed in E. coli or with Bm-103 and Bm-RAL-2 co-administered separately resulted in improved efficacy with consistent and significant 49%–51% and 56%–61% worm reduction at 90 days postinfection, respectively, as compared to alum adjuvant control. Immunization with both antigens (fusion or concurrent) also significantly reduced the fecundity of female worms (Arumugam et al., 2016). Both Bm-103 and Bm-RAL-2 induced elevated levels of antigen-specific IgG. The Bm-CPI-2 in its native form did not induce worm reduction but instead caused the relocation of filarial worm to the heart and lung (Arumugam et al., 2014b). However, after the functional Asn66 was mutated to Lys66, the mutant form of Bm-CPI-2, Bm-CPI-2M, significantly reduced worm burden (48.0%) as well as fertility of the female worms 90 days post challenge with B. malayi L3. The anti-Bm-CPI-2M sera were able to induce the killing of B. malayi L3s in vitro in the presence of gerbil peritoneal exudate cells, suggesting antibody-dependent cell-mediated cytotoxicity as a putative protective mechanism (Arumugam et al., 2014a). The results in the B. malayi/gerbil model further supported the selection of Ov-RAL-2, Ov-103, and Ov-CPI-2M as the most promising vaccine candidates for onchocerciasis, and Bm-103, Bm-RAL-2, and Bm-CPI-2M as the vaccine candidates for lymphatic filariasis.

Ov-103 and Ob-RAL-2 are down-selected as the leading vaccine candidates for onchocerciasis for further preclinical development. Based on the down-selection criteria highlighted above, these antigens exhibit the following properties: (i) immunodominant antigens recognized by putatively immune sera (Gallin et al., 1989, Lustigman et al., 1992); (ii) larval surface-associated antigens with immunological accessibility (Gallin et al., 1989, Lustigman et al., 1992); (iii) both antigens being immunogenic, inducing strong IgG responses in immunized mice; (iv) the significantly reduced L3 survival rate in mice immunized with recombinant Ov-103 and Ov-RAL-2 alone and in combination (Hess et al., 2014); (v) antibody inducing inhibition of L3 molting (George et al., 2019) or killing of nodular microfilariae in vitro (Lustigman et al., 1992); and (vi) protective homologues identified in other filaria (Pandiaraja et al., 2010; Arumugam et al., 2016), Ascaris (Wei et al., 2017), and hookworm (Zhan et al., 2004; Fujiwara et al., 2007).

Alum-formulated recombinant Ov-103 and Ov-RAL-2 induced significantly protective immunity against L3 challenge in diffusion chambers; however, the protective level remains partial and limited. To enhance the immunological responses and improve the protective immunity, recombinant Ov-103 and Ov-RAL-2 were formulated with different adjuvants that are approved for human use and known to induce dominant Th1, Th2, or combined Th1/Th2 responses including alum, Advax 1, Advax 2, CpG oligonucleotide (CpG), and MF59. Mice immunized with different adjuvant-formulated Ov-103 or Ov-RAL-2 individually or concurrently (combination) demonstrated different Th1/Th2 immune response profiles. Without adjuvant, recombinant Ov-103 and Ov-RAL-2 themselves did not induce any protective immunity. In the adjuvant groups, only alum-, Advax 2-, and MF59-formulated Ov-103 and Ov-RAL-2 induced significant protective immunity with similar levels of larval killing and host protection. The protection was associated with Th2-biased immune responses with IgG1 as the dominant antibody (Hess et al., 2016). Co-administration of Ov-103 and Ov-RAL-2 with the three adjuvants induced similar or even higher larval killing and host protection compared to mice receiving a single antigen. The highest level of larval killing was 47% achieved in mice immunized with two antigens formulated with Advax 2 as the adjuvant, suggesting that both antigens are not immunologically competitive and confer synergistic immune responses (Abraham et al., 2021). Based on chemokine profile and levels, more neutrophils and eosinophils were involved in the protective immune response induced by Ov-103, and more macrophage and neutrophil responses were involved in Ov-RAL-2-induced immune responses (Hess et al., 2016). Both recombinant Ov-103 and Ov-RAL-2 were very immunogenic and induced high levels of antigen-specific IgG without the induction of antigen-specific IgE responses. This is important as it may prevent the induction of an allergic reaction during immunization, which has hindered the continued vaccine development for Na-ASP-2, a potential vaccine candidate for hookworm infection that failed in clinical trial due to its ability to induce IgE in vaccinated people (Diemert et al., 2012).

The role of humoral immune responses in the protective immunity induced by immunization with Ov-103 or Ov-RAL-2 was confirmed in AID^-/- mice that was deficient in producing IgG antibody. AID^-/- mice immunized with Ov-103 or Ov-RAL-2 could not produce protection against challenge of O. volvulus L3. Further investigation on the IgG isotypes revealed that most people who lived in endemic areas and developed with putatively immune (86%) or concomitant immunity (95%) contained anti-Ov-103 and anti-Ov-RAL-2 cytophilic antibody responses (IgG1 and IgG3). Monospecific human anti-Ov-103 and anti-Ov-RAL-2 antibodies inhibited the molting of L3 when incubated with naive human monocytes (70%–80%) and to a lesser degree in the presence of naïve human neutrophils (46% for anti-Ov-103). Moreover, the inhibition of L3 molting with anti-Ov-103 antibodies was partially dependent on contact with the monocytes, while the inhibition of molting with anti-Ov-RAL-2 antibodies required the complete contact with the monocytes, suggesting that the mechanisms by which the two antibodies cause inhibition of molting are distinct. Notably, the L3 killing in Ov-103- and Ov-RAL-2-vaccinated mice only occurred when immune cells enter the chamber where the parasites reside, suggesting that ADCC is involved in the protective immunity induced by Ov-103 and Ov-RAL-2, and balanced Th1/Th2 responses are essential for the protective immunity to O. volvulus (George et al., 2019).

Before being advanced to clinical vaccine trials in humans, it was important to consider whether diverse host genetics may affect vaccine efficacy. Eight different genetically diverse collaborative cross recombinant inbred intercross mouse lines (CC-RIX) were immunized with both Ov-103 and Ov-RAL-2 formulated with the adjuvant Advax 2. After challenge, significant reductions in parasite survival were observed in 7 out of 8 tested CC-RIX lines with a similar level of protection as BALB/cByJ mice. However, there were no statistically significant correlations between larval killing and specific immune cell types, either with the levels of cytokines or chemokines measured in the chamber fluid and or in culture media of ex vivo antigen-stimulated spleen lymphocytes. Moreover, although immunization with both Ov-103 and Ov-RAL-2 induced antigen-specific IgG1 and IgG2a/b/c in BALB/cByJ and most of CC-RIX lines with different titers, there were no statistically significant correlations between antigen-specific antibody titer and the level of larval killing. These results reveal that the Ov-103 and Ov-RAL-2 vaccines are able to induce protection against Onchocerca L3 infection using varied mechanisms and in a wide range of host genetic backgrounds, suggesting that the vaccines could be translated for clinical development and may protect diverse human populations from infection with O. volvulus (Ryan et al., 2021).

In 2015, an international consortium including the Texas Children’s Center for Vaccine Development and its partners in US, Europe, and Africa launched The Onchocerciasis Vaccine for Africa Initiative (TOVA) with the goal of developing and evaluating an onchocerciasis vaccine for Africa (Hotez et al., 2015). Intense laboratory down-selection led to the selection of two antigens, Ov-103 and Ov-RAL-2, for further clinical development based on their consistent protective efficacies in mouse model studies. Although recombinant Ov-CPI-2M with functional Asn66 mutated to Lys66 induced a similar level of protection against O. volvulus L3 challenge, it was down-selected because of its ability to induce IgE responses in children ≤5 years of age. In comparison, there were no IgE responses found in Ov-RAL-2 and Ov-103 immunization (Abraham et al., 2021).

Currently, TOVA is moving forward to a preclinical trial in Cameroon for the immunogenicity and vaccine efficacy of co-administered Ov-RAL-2 and Ov-103 in cattle exposed to natural infection of O. ochengi (Makepeace et al., 2015). Due to the similarity of the life cycle transmitted by the same fly vector, overlapped endemic regions, and highly related vaccine candidates between O. volvulus in human and O. ochengi in cattle, the natural infection of cattle with O. ochengi in endemic areas is selected as the only one field vaccine trial model for validating the vaccine efficacy for human onchocerciasis (Makepeace and Tanya, 2016). Vaccination against onchocerciasis in natural host–parasite relationships represents the real-world vaccine efficacy in endemic regions. The data from the cattle trial in endemic regions will be translated to a phase I clinical trial in human. The target product profiles (TPPs) for developing an onchocerciasis vaccine for African living in endemic areas include the co-administration of Advax-2-adjuvanted Ov-103 and Ov-RAL-2 in children 1–5 years old (Hotez et al., 2015). However, a safety trial will be done in adults living in non-endemic and then in endemic regions to ensure that the vaccine is safe enough before it is tested in children. The aim of this vaccine is to prevent the establishment of infection in children before exposure to the infection. A serological investigation found that all 73 children 1–5 years old from a highly endemic region in Ghana did not show any anti-Ov-103 and Ov-RAL-2 IgE responses, while 3/27 and 1/27 of the children 6–8 years old displayed anti-Ov-103 or Ov-RAL-2 IgE responses, suggesting that continuous exposure to infective larvae in children over 5 years of age may develop IgE response to native Ov-103 and/or Ov-RAL-2 proteins during infection (Abraham et al., 2021). Therefore, the Ov-103 and Ov-RAL-2 vaccines should be safe for targeting children under 5 years of age without concern of pathological atopic responses as was other helminth vaccines when tested in humans (Hamilton et al., 2010; Diemert et al., 2012).

After decades of unremitting efforts by researchers around the world, especially by the close collaboration among the New York Blood Center (Dr. Sara Lustigman), Thomas Jefferson University (Dr. David Abraham), and Texas Children’s Hospital Center for Vaccine Development (Dr. Peter Hotez and Maria Elena Bottazzi), two antigens Ov-103 and Ov-RAL-2 have been selected for a bivalent vaccine for clinical efficacy trials based on their consistent protective efficacy induced in mice. This bivalent vaccine has been moved forward to a clinical trial in naturally infected cattle in Cameroon with the exciting endorsement of TOVA. Efficacy results from the cattle clinical trial with adjuvanted bivalent Ov-103 and Ov-RAL-2 vaccines will provide critical information for their immunogenicity and protective efficacy on worm burden and patency under natural infection conditions. If their protective efficacy is confirmed in the cattle clinical trial, the first bivalent onchocerciasis vaccine with Advax-2- or alum-formulated bivalent Ov-103 and Ov-RAL-2 will be produced in large scale under cGMP conditions and used to perform a phase I safety clinical trial. The ultimate goal is to develop a vaccine that can be used safely in children under 5 years of age in endemic regions that prevents infection with O. volvulus and protects them from developing adult worm pathological consequences with time.

While we are testing our first generation of the onchocerciasis vaccine, the co-administration of recombinant Ov-103 and Ov-RAL-2 adjuvanted with Montanide™ ISA 206VG in cattle in a preclinical trial in Africa, there still are some challenges. Notably, the protection rate of current selected Ov-103 and Ov-RAL-2 against Onchocerca L3 challenge is partial and limited despite the intensive search for the appropriate adjuvants and immunization regime to induce better protection. We will need to continue modeling whether the partial protection rate of current selected Ov-103 and Ov-RAL-2 against Onchocerca L3 challenge will be sufficient to support elimination (Turner et al., 2015). We also need to make more efforts to enhance their immunogenicity and vaccine efficacy by (i) searching and identifying the optimal adjuvant and formulation that might induce better protective immunity in humans; (ii) optimizing the co-administration of both antigens by making a fusion protein or combination of both individual antigens, to reach better protective efficacy; (iii) increasing efforts for the discovery of additional effective vaccine candidates, including new-generation vaccine antigens identified using new technologies such as functional genomics, transcriptomic, proteomic, secretomics, immunomics, and the mRNA vaccine technology; (iv) developing novel diagnostic assays that can support the rapid monitoring of vaccine efficacy in human clinical trials; (v) developing suitable product development strategies and the preferred target product profile; (vi) enhancing the collaboration between laboratory researchers and local health workers; (vii) promoting financial support from international organizations and/or governments to bring such a vaccine toward clinical trial and licensure; and (viii) advocating integration of such a vaccine with other elimination programs, including South-Sahara Africa government agencies in such efforts.

BZ conceived and drafted the manuscript. SL, PH, and MB revised and added insight into the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by NIH/NIAID R01AI078314.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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