Plant-Derived Human Vaccines: Recent Developments

The first plant-produced vaccine that was approved by the United States Department of Agriculture (USDA) for commercial use was a veterinary vaccine in 2006 against Newcastle disease virus (NDV) which affects chickens [74]. However, although the discovery that vaccines could be produced using a plant platform materialised early on, the development of vaccine production in plants has been slow and no plant-produced human vaccines have been licensed to date. The concept underwent a lapse due to lack of regulatory frameworks and an unproven track record in the industry [18]. Most early studies of vaccines have focussed on optimising expression and increasing yields as well as developing purification protocols rather than targeting commercial competitiveness, as requirements for clinical trials are challenging and are fraught with more stringent regulatory requirements [75]. However, over time, the consolidation of protocols, establishment of GMP-compliant plant production facilities, drafting of regulatory frameworks as well as techno-economic analyses [76,77,78] demonstrating feasibility and commercial applicability have established the production of vaccines in plants as a significantly more accepted enterprise [18]. A number have been tested in clinical trials [79,80,81] and the first plant-produced vaccine for human use against COVID-19 was recently authorised by Health Canada [20] after the successful outcome of phase III clinical trials [81]. Below, we discuss some of the more recent developments in the last 5 years (2018–2022) of plant-produced human vaccine research against various disease targets (Table 1).

Table 1 Examples of recently developed human vaccines in plants (within the last 5 years)Hepatitis B Virus (HBV)

The first human vaccine candidate to be produced in plants was for hepatitis B virus (HBV) in transgenic tobacco (N. tabacum) [82]. The expression of HBV surface antigens (HBsAg) resulted in the assembly of particles resembling those from human-derived serum and produced recombinantly in yeast, heralding the potential for plant-made vaccines. Despite successful production of HBV VLPs in both potato and lettuce, their testing in phase I clinical trials [83, 84], the production of HBV VLPs transiently in tobacco [85] and increased levels of expression [86], commercialisation of this vaccine has still not been pursued. In the last 5 years, more recent studies have looked at the use of HBcAg expression in plants as a therapeutic vaccine [87]. Pyrski et al. [88] have recently reported the parenteral immunisation of mice with HBcAg produced transiently in N. benthamiana with an oral boost of HBcAg-expressing lettuce. The low dose elicited a specific high titre antibody response with a predominant Th1 response and evidence of a Th2 response after the oral boost.

Hepatitis E Virus (HEV)

The ORF2 AA551-AA607 immunogenic epitope of the hepatitis E capsid protein was inserted into one of the HBcAg surface loops using the Tandem Core Technology [46] and expressed transiently in N. benthamiana. Chimaeric VLPs ranging in size from 28 to 38 nm in diameter were visualised by electron microscopy, which had ‘knobbly’ surfaces, most likely representing the epitopes being displayed on the HBcAg surface spikes. Although no animal immunology has been carried out yet, the chimaeric VLPs showed they could bind both to an anti-HBcAg mAb as well as swine serum containing anti-HEV IgG, suggesting a possible bivalent vaccine function for the candidate [89].

Poliovirus

Marsian et al. [42] have reported the development of poliovirus VLPs in N. benthamiana by co-expression of a mutated capsid sequence of poliovirus serotype 3 (PV3) and the poliovirus 3CD protease which cleaves the intermediate capsid sequence P1 into three capsid proteins (VP0, VP1 and VP3), which assemble into VLPs. These were shown to retain the stability and potency of the currently used inactivated polio vaccine (IPV). The vaccine was shown to protect immunised transgenic mice expressing the human poliovirus receptor after challenge with the wild-type Saukett strain of PV3. More recently, poliovirus Sabin subunit capsid-forming proteins (VP1, VP2, VP3 and VP4) were produced individually in transgenic N. tabacum plants. Immunisation of mice with four subcutaneous injections 2 weeks apart and four subsequent oral immunisations with suspensions of lyophilised transgenic leaf material containing all four proteins induced local humoral immune responses, exhibiting potential for further application [61].

Flaviviruses

Ponndorf et al. [90] have recently reported the successful production of dengue virus serotype 1 (DENV1) VLPs in N. benthamiana. Co-expression of constructs encoding the DENV1 structural polyprotein C-pRM-E together with a construct encoding an NS5-truncated non-structural protein (NSP) domain of DENV1 resulted in particles ranging in size from 25 to 40 nm. Immunisation of mice with a prime-boost regimen of 10 µg VLPs per dose was shown to elicit anti-DENV1-specific antibodies.

Pang et al. [91] utilised the Tandem Core Technology previously developed using HBcAg display [46]. A consensus sequence representing four DENV serotypes of envelope protein domain III (EDIII) was inserted into one of the immunodominant c/e1 surface loop regions encoding a tandem HBcAg. VLPs displaying EDIII epitopes were transiently assembled in N. benthamiana after Agrobacterium-mediated infiltration and mice immunised with the VLPs showed some seroconversion.

Up until 2015, there have been a number of developments of subunit and VLP-based vaccine candidates produced in plants against West Nile virus (WNV), reviewed in detail by Chen [92]. Subsequently, He et al. [62] showed that soluble WNV EDIII expressed transiently in N. benthamiana induced a potent immune response in mice and was potentially protective in mice challenged with WNV [63]. The potential efficacy of EDIII was taken further by two different studies testing the immunogenicity of displaying EDIII on particles. He et al. [93] transiently expressed a construct encoding HBcAg fused to WNV EDIII which resulted in the assembly of VLPs resembling wild-type particles. Results from immunisation of mice demonstrated high levels of immunogenicity with potent anti-IgG-specific titers, equivalent to levels required for protection. Stander et al. [51] developed an antigen display vaccine for WNV. This vaccine candidate relied on the use of the SpyTag-SpyCatcher (ST/SC) technology [50]. The immunogenic WNV EDIII fused to SC was expressed transiently in N. benthamiana. Phage AP205 coat protein genetically fused to ST was similarly expressed in N. benthamiana and assembled into particles (Fig. 2a). Co-purification of these from N. benthamiana leaves resulted in AP205 particles displaying conjugated EDIII. Mice immunised with the AP205:EDIII particles elicited potent IgG responses.

Fig. 2figure 2

Examples of antigen-display particles and VLPs. a ST-fused phage AP205 particles displaying SC-fused WNV EdIII produced in N. benthamiana. b HPV VLPs produced in N. benthamina by expression of HPV L1 capsid protein. EdIII envelope protein domain III, HPV human papillomavirus, SC SpyCatcher, ST SpyTag, VLP virus-like particle, WNV West Nile virus

Similar to the WNV HBcAg-EDIII VLP, Yang et al. [94] showed that Zika virus (ZV) EDIII fused to HBcAg resulted in great VLPs when expressed transiently in N. benthamiana and elicited potent humoral and cellular responses in mice at levels exceeding those correlating with protective immunity against several Zika strains tested. A recently reported advancement has been the co-delivery of such particles with RICs. Based on previous research carried out to develop an Ebola virus subunit vaccine consisting of an Ebola glycoprotein GP1 fused to the C terminus of a cognate mAb heavy chain (HC), co-expression of matching light chain (LC) in N. benthamiana using a geminiviral vector was shown to form immunoglobulins. C1q binding assays and purification and analysis confirmed assembly of ICs in planta and immunisation of mice showed some immunogenicity [55]. Further work confirmed that co-administration of the RICs with adjuvant elicited a more robust immune response and protected mice challenged with Ebola virus [95]. More recently, the RIC platform was enhanced to accommodate a wider variety of antigens. Diamos et al. [57] showed that fusion of Zika EDIII to either the N or the C terminus of the HC of a Zika mAb showed immunogenicity in mice, which was enhanced further when co-delivered with HBcAg displaying Zika EDIII.

Currently, all yellow fever virus (YFV) vaccines licensed for use are live attenuated virus vaccines produced in eggs; for example, YF-Vax® (Sanofi-Pasteur, Lyon) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro). Recently, Tottey et al. [96] developed YFV vaccine candidates through transient expression of the envelope protein alone (YFE) or fused to the bacterial enzyme lichenase (YFE-LicKM) in N. benthamiana. High virus-neutralising titers were observed following immunisation in mice for both YFE and YFE-LicKM with 71% and 88% of animals surviving lethal YFV challenge, respectively. In addition, the YFE and YFE-LicKM vaccine candidates induce high virus-neutralising antibody responses in Macaque monkeys.

Rotavirus

Currently, available vaccines prequalified for clinical use by the World Health Organization against rotavirus comprise only live-attenuated virus preparations for oral use: RotaTeq® (Merck, Kenilworth), Rotarix™ (GlaxoSmithKline Biologicals, Rixensart), Rotavac® (Bharat Biotech, Hyderabad) and ROTASIIL™ (Serum Institute of India, Hadapsar) [97]. Kurokawa et al. [98], however, have developed a monovalent Ro-VLP vaccine composed of rotavirus surface proteins VP7, VP6 and VP2 of the rotavirus G1 genotype as well as the rotavirus non-structural helper protein NSP4. Agrobacterium-mediated transient expression in N. benthamiana yielded VLPs that were structurally similar to the triple-layered rotavirus particles. Rats immunised intramuscularly (IM) with two doses, 3 weeks apart, of aluminium hydroxide adjuvanted Ro-VLPs (1, 5 or 30 µg), elicited robust neutralising antibodies. Non-adjuvanted Ro-VLPs, at doses of 5 µg or 30 µg, also induced potent neutralising antibodies. The safety and immunogenicity of the vaccine was subsequently evaluated in a phase I trial (NCT03507738) in adults (aged 18–35 years), toddlers (12–24 months) and infants (6–10 weeks) [99] and was well tolerated with few mild adverse events in all cohorts. Anti-G1P IgG responses and neutralising antibodies were elicited in infants regardless of dose levels (7 µg or 21 µg adjuvanted).

Norovirus

Diamos and Mason [100] reported the assembly of human norovirus VLPs by transient expression of the GI or GIII.4 capsid protein in N. benthamiana using a geminiviral vector. The VLPs were purified up to 90% but no immunogenicity or efficacy of the vaccine candidate has been reported to date by this group. A noteworthy development by another group has reported the co-administration of insect cell-produced rotavirus VP6 with transiently plant-produced GI.3 and GII.4 VLPs in mice, which generated norovirus-specific antibodies against the vaccine as well as heterologous norovirus genotypes [101]. Norovirus VLPs alone did not elicit any response and it was concluded that the rotavirus VP6 had an adjuvant effect as doses as low as 1 µg were effective in stimulating specific antibodies. A bivalent VLP norovirus vaccine candidate comprising the GI.4 and GII.4 antigens (rNV-2v) has been produced transiently in N. benthamiana [102]. Results obtained from preclinical toxicity studies in rabbits illustrated the safety and tolerability of the vaccine. In addition, strong rNV-2v-specific antibody responses were observed in both male and female rabbits in the absence of adjuvants with very high levels of blocking antibodies.

Rabies virus (RABV)

Since 1967, the standard rabies vaccine used has consisted of the attenuated-type human diploid cell vaccine [103], with several other attenuated and inactivated RABV vaccines approved for human use. Due to the cost associated with the production of these vaccines in animal cell culture, Mammadova et al. [64] recently investigated the efficacy of a subunit RABV vaccine produced in N. benthamiana. Following transient expression of a truncated form of the G protein (RG2), a yield of ~ 32 mg/kg fresh leaf weight of purified RG2 protein was obtained and high RG2-specific antibody titers were observed in mice following IM immunisation with two doses of 5 µg RG2 with Alhydrogel®.

Bacillus anthracis (Anthrax)

Currently, there is only one anthrax vaccine approved for use in the US, namely BioThrax® (Emergent BioSolutions, MD, USA), which is prepared from cell-free filtrates of microaerophilic cultures of an avirulent, nonencapsulated strain of Bacillus anthracis and formulated with an aluminum hydroxide adjuvant. Recombinant subunit vaccine research against anthrax has been focused on the protective antigen (PA) which is the principal virulence factor of B. anthracis. In 2013, a full-length PA (pp-PA83) prophylactic vaccine candidate transiently produced in N. benthamiana was shown to elicit high neutralising antibody titers in mice and rabbits when administered with Alhydrogel® [65]. Additionally, rabbits immunised with the pp-PA83 vaccine (with Alhydrogel®) were protected from a lethal aerosolised B. anthracis challenge. Subsequently, very recently, a phase I trial (NCT02239172) in healthy adults (18–49 years) was conducted with pp-PA83, named PA83-FhCMB vaccine candidate [66]. There were 30 participants across four dose-escalating vaccine groups, 12.5 µg (n = 5), 25 µg (n = 5), 50 µg (n = 10) and 100 µg (n = 10), that received three IM doses of PA83-FhCMB with Alhydrogel®. The plant-made vaccine was found to be safe and well tolerated in all groups with no observed serious adverse events. In addition, all participants seroconverted by day 56, with the highest geometric mean titre occurring by day 84 following the third dose. The authors concluded that a fourth booster dose of the PA83-FhCMB vaccine at 6 months could be beneficial since an increase in the geometric mean concentration was observed for individuals who received the BioThrax® vaccine following a fourth booster dose.

Malaria

In the past 15 years, a plant-based malaria transmission blocking vaccine (TBV) against the Plasmodium falciparum protein, Pfs25, has been in development. The TBV comprises the Pfs25 protein fused to the Alfalfa mosaic virus coat protein, and transient expression in N. benthamiana using a TMV-based hybrid vector resulted in the formation of a chimaeric non-enveloped VLP (Pfs25-CP VLP) [104]. Approximately 20–30% of the Pfs25 protein was incorporated onto the VLP surface with a diameter of ~ 19 nm [105]. Mice immunised IM with two doses of Pfs25-CP VLPs at either 1.0 or 0.1 mg (equivalent to Pfs25) with Alhydrogel® had high IgG responses with 100% transmission blocking (TB) activity that was maintained through day 168 post-immunisation. The immunogenicity study was repeated in mice and similar IgG responses were observed and maintained for 5 months. As observed in the first immunogenicity study, a TB activity of 100% following the booster dose was obtained and maintained at 98–99% through day 168. A third mouse immunogenicity study with a single Pfs25-CP VLP IM dose at either 0.2, 1.0, 5.0 or 25 mg (equivalent to Pfs25) with Alhydrogel® was performed and high Pfs25-specific IgG titers were observed and maintained for the 6-month test period. For doses ≥ 5.0 mg, 100% TB activity was observed, while 99.5% and 98.8% was observed for doses of 1.0 and 0.2 mg, respectively. Remarkably, for doses ≥ 1.0 mg, the TB activity was maintained at ~ 94% for 6 months post-vaccination.

These results highlighted the potential of Pfs25-CP VLP as a malaria TBV and subsequently Chichester et al. [106] produced a candidate vaccine named Pfs25 VLP-FhCMB in N. benthamiana under Good Manufacturing Practice guidelines for a phase I trial (NTC02013687) in adults (aged 18–50 years). The Pfs25 VLP-FhCMB (with Alhydrogel®) dose groups consisted of 2 µg (n = 6), 10 µg (n = 6), 30 µg (n = 16) and 100 µg (n = 16) total protein that was administered IM three times. The plant-made Pfs25 VLP-FhCMB vaccine was shown to be safe in healthy adults with no vaccine-related serious adverse events or dose-limiting or dose-related toxicity; this highlighted the safety and tolerability of the vaccine. Good antibody responses were observed in individuals immunised with vaccine doses > 30 µg, however the transmission-reducing activity of the generated antibodies was weak. The authors suggested that the low transmission reducing activity might be overcome with the use of alternative adjuvant formulations.

Human Papillomavirus (HPV)

Although there are currently very efficacious HPV VLPs on the market made in both yeast (Gardasil®, Merck, Whitehouse Station) and insect cells (Cervarix™ ,GlaxoSmithKline Biologicals, Rixensart) [107], there has been ongoing research since 2003 into the production of plant-produced HPV VLPs by transient expression of L1 capsid protein in plants (Fig. 2b) (extensively reviewed elsewhere [40]). Further developments on this include the development of chimaeric HPV-16 VLPs consisting of substituted L2 peptides into four different regions on the DE surface loop of HPV-16 L1 or at its C-terminal region [43]. The former resulted in complete assembly of VLPs while for the latter, assembly was partial, resulting in pentavalent capsomeres. Immunogenicity studies on mice immunised with a cocktail of these resulted in elicitation of neutralising antibodies to HPV-16 as well as cross-neutralisation with HPV-58 and -18.

Influenza Virus

Prior to the emergence of the COVID-19 pandemic, the influenza vaccine developed by Medicago Inc. (Québec, Canada) was the vaccine most likely to put plant-produced vaccines on the commercial map. In 2008, D’Aoust et al. [108] showed that individual expression of the HA protein of two strains (H5/N1 and H1/N1) of influenza A expressed transiently in N. benthamiana assembled into VLPs. These consisted of pleomorphic enveloped particles of ~ 100 nm in diameter, studded with trimeric HA spikes. Latterly, cryo-electron microscopy and tomography have confirmed the ovoid or discoid structure of the VLPs bearing HA trimers evenly distributed at their surface or presented on their outer diameter, respectively [109]. In addition, the VLPs are morphologically stable for at least 12 months at 4 °C. Immunisation of mice with H5 VLPs demonstrated immunogenicity (in the form of HI antibodies) against homologous virus and complete protection with a heterologous H5/N1 challenge.

Further studies showed that VLPs could be made similarly for other influenza HA strains including H2, H3, H6 and H9 as well as influenza B HA [110]. Immunisation of ferrets—the target animal model for influenza [111]—with the VLPs showed good protective responses against heterologous challenges, fulfilling the criteria of the European Committee of Medicinal Products (CHMP) for human use [112]. A phase I preclinical and safety trial in humans (NCT00984945) of H5N1 VLPs demonstrated promising immunogenicity and a good safety profile [113]. This encouraged Medicago Inc. to develop a VLP platform using tobacco plants as the production host after demonstrating that they could produce more than 10 million doses of influenza H1N1 VLP vaccines in 1 month, as part of a DARPA Blue Angel program in 2012 [114]. Landry et al. [115] showed in a phase I trial (NCT01302990) that unadjuvanted H1N1 and H5N1 plant-produced VLPs (5-, 13- or 28-µg doses) elicited T-cell responses in humans that were durable and cross-reactive. Pillet et al. [116] further illustrated in a phase I–II clinical trial (NCT01991587) that a quadrivalent VLP (QVLP) vaccine comprising influenza A H1N1 and H3N2, and influenza B Yamagata and Victoria lineage HA VLPs (3, 9 or 15 µg) was safe, met the HI antibody titers of the European licensure criteria and also induced and sustained a substantial CD4+ T-cell response which was cross-reactive against a different H3N2 strain and B lineage. A phase II dose-ranging trial of humans (NCT01991561) immunised with H5N1 VLPs co-administered with GLA-SE adjuvant showed that lower doses (3.5 or 7.5 µg) than those previously tested could be used whilst still meeting the licensure criteria for HI antibody responses as well as producing a sustained polyfunctional and cross-reactive CD4+ T-cell response [117]. These results bode well for dose-sparing requirements during a pandemic.

However, a further two phase II trials (NCT02233816 and NCT02236052) reported by Pillet et al. [118] to test and compare the effect of doses on older adult populations confirmed that a 30-µg dose of QVLPs provided the most consistent humoral and cellular immunity in the two age groups tested (18–49 years and ≥ 50 years) and this dosage was agreed upon as the best compromise for all age groups for further clinical trials. During the 2017–2018 and 2018–2019 influenza seasons, two randomised phase III trials (NCT03301051 and NCT03739112) were carried out by Medicago Inc. to test safety, immunogenicity and efficacy of a QVLP vaccine (non-adjuvanted 30-µg dose) in adults (18–64 years) and older adults (≥ 65 years), respectively [119]. These trials are now complete, both studies reporting elicitation of strong CD4+ T-cell responses to the QVLP vaccine.

In 2020, British American Tobacco plc (UK) and Kentucky Bioprocessing Inc (USA) (BAT/KBP) announced the approval for registration of a phase I trial (NCT04439695) to test the immunogenicity and safety of a quadrivalent influenza vaccine produced in plants (KBP-V001). Details of the vaccine candidate are not published, but it is reported to make use of the same plant-based technology platform as described for SARS-CoV-2 in the following section [120].

SARS-CoV-2

The COVID-19 pandemic starting in 2019, triggered considerable efforts to develop vaccines against its causative agent, SARS-CoV-2. It proffered an ideal opportunity for the biopharming community to prove that plants are a model platform for vaccine production under pandemic circumstances [121,122,123,124]. The platform’s ability to develop a candidate in a short time frame and rapidly scale up the production of vaccine candidates should fulfil all the requirements imposed by the pandemic and there have been several reports on COVID-19 vaccine developments.

Having completed their phase III influenza trials, Medicago Inc. were able to rapidly adapt their technology to develop a VLP vaccine similar to that reported for influenza, using a modified spike (S) glycoprotein from SARS-CoV-2 strain hCoV-19/USA/CA2/2020 [125]. The transmembrane (TM) and cytoplasmic tail (CT) regions of SARS-CoV-2 S were substituted with those of the influenza HA strain previously used [108], which increased VLP assembly and budding. In addition, several substitutions in S were made to increase stability at the S1/S2 cleavage site and stabilise the protein in a pre-fusion conformation. The coronavirus-like particle (CoVLP) vaccine was tested in a phase I randomised trial of 180 adults (NCT04450004) who were immunised IM with two doses ranging from 3 to 15 µg either alone or adjuvanted with ASO3 or CpG1018. Interim data 21 days after the second boost reflected neutralising antibody titers as well as IFN-γ and IL-4 cellular responses which were not affected by different doses, but which increased significantly with either of the adjuvants used. However, ASO3 was more effective than CpG1018 in enhancing the immune response as well as dose sparing. The safety profile was similar to that reported for Medicago’s plant-produced influenza QVLP vaccine [126], with adverse events (AEs) more prevalent in patients who received adjuvanted doses and those receiving CoVLP alone showing few AEs.

Medicago Inc. subsequently followed up with a phase II/III trial to assess the immunogenicity, safety and efficacy of the CoVLP vaccine formulation and dosing regimen (NCT04636697). Most encouragingly, results show that the vaccine had 78.8% efficacy against moderate-to-severe disease and a 74% efficacy against seronegative participants [81]. Moreover, the vaccine elicited 69.5% efficacy against COVID-19 caused by five different variants including those of delta, gamma, alpha, lambda and mu. In February 2022, it was officially approved for human use in Canada by Canada Health and is to be marketed under the name of COVIFENZ® (Medicago Inc., Québec) [20, 127].

Kentucky Bioprocessing Inc, the US biotech subsidiary of BAT, together with several others have also developed a SARS-CoV-2 vaccine candidate (CoV-RBD121-NP) by using a previously developed virus-like particle-type nano-particle (VLP-type NP) vaccine platform [47] to produce a TMV-like NP displaying a chemically conjugated SARS-CoV-2 antigen [128]. The antigen—CoV-RBD121—comprised amino acids 331–632 of the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) fused to a human IgG1 Fc domain to enhance stability of the RBD as well as facilitate ease of purification. CoV-RBD121 was transiently expressed in N. benthamiana plants, purified and then chemically conjugated to modified TMV particles, also produced in plants by infection of N. benthamiana with virions using a previously established method [129]. Immunogenicity of CoV-RBD121-NP was tested in mice immunised subcutaneously with two doses 2 weeks apart; two different vaccine doses were tested (15 or 45 µg), with or without 7909 CpG adjuvant. Overall, antibody responses were strong irrespective of whether formulated with or without 7909 CpG. Although neutralising antibodies were stimulated, titers were much higher for the higher dose of 45 µg used, irrespective of the presence or absence of the adjuvant. Most importantly, characterisation of the NPs and stability assays showed that this vaccine candidate was stable for up to 12 months at 2–8 °C as well as 22–28 °C, fulfilling an important criterion for worldwide distribution requirements for vaccines. In addition, the pilot batch of CoV-RBD121-NP was manufactured within 28 days of receiving the SARS-Cov-2 RBD sequence showing that the platform can adapt rapidly to vaccine target demands in the event of a pandemic [128]. BAT/KBP has recently been given approval to commence with a phase I clinical trial to test its safety and immunogenicity (NCT04473690).

In 2021, Siriwattananon et al. [68] reported the production of a SARS-CoV-2 subunit vaccine by transient expression in N. benthamiana; the vaccine comprised SARS-CoV-2 RBD fused to the human IgG1 Fc fragment. The plant-made RBD-Fc fusion protein with alum elicited high neutralising antibody titers in mice and cynomolgus macaques following two IM doses of 10 µg and 25–50 µg, respectively. The vaccine candidate was evaluated further with attention to adjuvant formulation effects on the vaccine immunogenicity [67]. Four commercial adjuvants—Alhydrogel®, monophosphoryl lipid A from Salmonella Minneso

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