Preliminary Evaluation of the Safety and Immunogenicity of an Antimalarial Vaccine Candidate Modified Peptide (IMPIPS) Mixture in a Murine Model

Malaria continues being a high-impact disease regarding public health worldwide; the WHO report for malaria in 2018 estimated that ~219 million cases occurred in 2017, mostly caused by the parasite Plasmodium falciparum. The disease cost the lives of more than 400,000 people, mainly in Africa. In spite of great efforts aimed at developing better prevention (i.e., a highly effective vaccine), diagnosis, and treatment methods for malaria, no efficient solution to this disease has been advanced to date. The Fundación Instituto de Inmunología de Colombia (FIDIC) has been developing studies aimed at furthering the search for vaccine candidates for controlling P. falciparum malaria. However, vaccine development involves safety and immunogenicity studies regarding their formulation in animal models before proceeding to clinical studies. The present work has thus been aimed at evaluating the safety and immunogenicity of a mixture of 23 chemically synthesised, modified peptides (immune protection-inducing protein structure (IMPIPS)) derived from different P. falciparum proteins. Single and repeat dose assays were thus used with male and female BALB/c mice which were immunised with the IMPIPS mixture. It was found that single and repeat dose immunisation with the IMPIPS mixture was safe, both locally and systemically. It was observed that the antibodies so stimulated recognised the parasite's native proteins and inhibited merozoite invasion of red blood cells in vitro when evaluating the humoral immune response induced by the IMPIPS mixture. Such results suggested that the IMPIPS peptide mixture could be a safe candidate to be tested during the next stage involved in developing an antimalarial vaccine, evaluating local safety, immunogenicity, and protection in a nonhuman primate model.


Introduction
Malaria represents one of the greatest public health problems worldwide. According to the World Health Organization (WHO),~219 million new malaria-related cases occurred in 2017 accompanied by~435,000 deaths. The African continent was the most affected region in the world (92% of cases and 93% of deaths) [1]. The Global Technical Strategy for Malaria 2016-2030 (WHO) has suggested reducing malarial incidence and mortality by at least 90% and eliminating it in at least 35 countries by 2030 through prevention, diagnosis, and treatment strategies [2].
No significant progress has been observed to date regarding the reduction of cases of malaria worldwide despite the differing strategies used for combating this disease (using insecticide-impregnated mosquito nets for controlling the vector, chemoprophylaxis, and case management) [1,2]. The most recurrent problem is concerned with the increase in strains which are resistant to antimalarial drugs and insecticide-resistant mosquitoes; this has necessitated the development and combined use of new control and prevention methods, especially a vaccine having high protection capability as time elapses [1,3].
Nuclear magnetic resonance (NMR) and in silico structural binding studies have shown that mHABPs having polyproline II (PPIIL) helix structures [23,24] can bind to HLA-DRβ1 * molecules covering most MHC-II allele variants [21,25,26] and have greater interaction with the T cell receptor (TCR). This would suggest the stable formation of the MHC-II-mHABP-TCR trimer complex and thus the capability for inducing a protective immune response [27][28][29][30][31]. Protection-inducing mHABPs have thus been called Pf immune protection-inducing protein structures (IMPIPS) in view of their close structure-protection relationship [32,33].

Peptide Synthesis and Purification.
Twenty-three polymer peptides (Table 1) were modified following previously reported principles [32,33,40,41] to render them immunogenic and then synthesised using solid-phase multiple peptide synthesis following the tert-butyloxycarbonyl (t-Boc) synthesis strategy described by Merrifield [42] and modified by Houghten [43]. All peptides were derived from fully conserved and functionally relevant regions of the corresponding proteins. Such a Merck-Hitachi L-6200 A chromatograph (Merck) fitted with a UV-VIS L-4250 210 nm wavelength detector was used for determining synthesised peptide purity by high-performance reversed-phase liquid chromatography. A Microflex mass spectrometer (Bruker Daltonics) was then used for characterising them by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF).

Animals.
Forty BALB/c mice (20 males and 20 females) were used for evaluating the formulation's safety and immunogenicity. The mice were aged 5 to 6 weeks when they were immunised, according to WHO recommendations [37]. The mice were acquired from the Universidad Nacional de Colombia's Faculty of Animal Science's Biotherium.

Single Dose Local
Tolerance. This assay enabled determining possible inflammatory reactions at the different treatments' inoculation sites [37]; this involved using 18 BALB/c mice (9 males and 9 females), following the protocols established by the regulatory authorities [34][35][36][37]. The animals were randomly assigned to 3 groups (Table 2), each consisting of 3 males and 3 females, in line with the principles of reduction and refinement [44].
The animals were immunised by subcutaneous (SC) route at the base of the tail with 100 μL of the formulation. The animals were observed twice a day after they had been immunised for detecting changes in their behaviour, signs of disease, or toxicity. The injection site and the tissue around it were examined 1, 3, 24, 48, and 72 hours after immunisation to ascertain the presence of erythema, oedema, eschar, and necrosis; the parameters described by Cox were used for evaluating their degree [45]. The animals were anesthetised with ketamine (80-120 mg/kg) and xylazine (5-16 mg/kg) by intraperitoneal (IP) route on the third day and sacrificed by cervical dislocation.

Repeat Doses.
Twenty-two BALB/c 5-to 6-week-old mice (11 males and 11 females) were randomly distributed into three groups for evaluating possible toxic reactions produced by repeat inoculations (SC) of the Pf-IMPIPS peptide mixture (30 μg in total). Toxicity due to repeat doses can occur as a result of repeat administration of a product over a specific period [35][36][37] (Table 3).
The animals were immunised 4 times with a 14-day interval as the amount of doses in an animal model must be equal to or greater than the amount of doses for clinical assays [37], and it would be expected that the amount of doses administered would not exceed two in clinical assays ( Figure 1).
Each group was immunised with 100 μL of the formulation on days 0, 14, 28, and 42; the formulation was administered by SC route at the base the tail. The immunisation sites were examined 1, 3, and 24 h after each injection looking for signs of erythema, oedema, eschar, and necrosis.
The animals were observed twice per day for evidence of any adverse reaction to the injection or the presence of disease, and a weekly physical examination was made for monitoring every animal's overall state of health. Their weight and food consumption were also monitored before beginning the immunisation protocol and after immunisation on days 0, 3, and 7 and every week thereafter until day 70.
Mouse body temperature was measured with an infrared thermometer (Benetech GM320) before and after each immunisation (0, 4, and 24 h) at five different sites on their abdomens. The average of five readings was recorded [46].
Blood samples were taken from the facial vein before immunisation and on days 1, 3, 40, 43, and 70 following the first immunisation to rule out acute and chronic alterations and in case of any abnormal findings. These samples were used for evaluating blood urea nitrogen (BUN), creatinine (CRE), haematocrit (HCT), red blood cell (RBC) count, white blood cell (WBC) count, and total plasma protein   (TPP) levels. The animals were anesthetised with ketamine (80-120 mg/kg) and xylazine (5-16 mg/kg) on day 70 by IP route and sacrificed by cervical dislocation [47]. The mice were necropsied, and kidney, heart, duodenum, spleen, and liver samples were taken for histological study to evaluate possible damage. The samples were kept in 10% formaldehyde and processed and analysed at the Universidad de Ciencias Aplicadas y Ambientales (U.D.C.A) pathology laboratory.
2.3. Immunogenicity 2.3.1. Indirect Immunofluorescence Assay (IFA). The indirect immunofluorescence assay (IFA) was used for evaluating an antibody's ability to recognise the parasite's native proteins, according to a previously reported methodology [48]. Briefly, RBC infected with mature schizont-stage parasites were taken from a P. falciparum FCB2 strain continuous culture synchronised with 5% sorbitol, at 5% to 7% parasitaemia. The RBCs were washed three times with phosphate-buffered saline (PBS) (7.2 to 7.4 pH) and spun at 1,200 g for 5 min. The pellet was suspended in filtered PBS until reaching 1% final dilution, 20 μL/well of this suspension was seeded on 8well slides and left to settle for 20 minutes, and the supernatant was collected. The slides were left to dry at room temperature (RT); they were then blocked with 30 μL/well PBS-1% skimmed milk for 10 min at (RT), washed once with PBS (7.2 to 7.4 pH) for 5 min, and left to dry.
Then, in duplicate, 2.5 μL/well serum from the final bleeding was seeded on 8-well slides at 1 : 20 dilution in PBS. The slides were incubated in a moist chamber for 30 min, washed with PBS (7.2 to 7.4 pH) six times for 5 min each wash, and left to dry. This was followed by placing 10 μL/well fluorescein isothiocyanate-(FITC-) labelled antimouse IgG (Vector Laboratories, Inc.) in each well at 1 : 20 dilution in PBS as well as 1 : 80 4 μL/well Evans blue to reduce background and increase contrast in the imaging/reading. The slides were incubated in a moist chamber in the dark for 30 min, washed, and left to dry. The slides were observed with a fluorescence microscope (Olympus B51) at 1,000x magnification. The assay was made by pooling all the sera from each group due to the low serum volume available. Serum from a P. berghei-infected mouse was used as the positive control. The immunofluorescence signal was analysed semiquantitatively in the photographs already taken as follows: (+++) corresponds to the maximum fluorescence (positive control), (0+) to the negative control, (++) to IMPIPS plus adjuvant, and (+) to IMPIPS plus PSS.

Invasion Inhibition
Assay. The P. falciparum FCB-2 strain (parasite ring stage) culture, previously synchronised with 5% sorbitol, 1-8 h postinvasion, was used for determining the ability of serum from the mice immunised with the IMPIPS mixture to inhibit Mrz invasion of erythrocytes [49]. A 384-well plate [50] seeded with 1.7 μL/well parasite culture (2% haematocrit and 0.1% parasitaemia) was incubated with 10 μL/well of serum from the final bleeding which had been inactivated (preimmune and postimmune) at 20%, 10%, 5%, and 2.5% concentrations (%v/v). Each well's final volume was 50 μL, which was completed with RPMI 1640 media (Gibco); each sample was analysed in duplicate. The plate was incubated at 37°C for 48 h in a 5% O 2 , 5% CO 2 , and 90% N 2 atmosphere. Parasitised RBCs (pRBCs) and sera from control group mice (day 0) were used as the negative control, whilst human RBCs with chloroquine (150 nM) were used as the positive control for the invasion inhibition assay. Parasite culture supplemented with healthy human plasma was used as the culture control. The plate was spun at 1,800 rpm after 48 h incubation, and the culture supernatants were removed; the cells were then labelled with 1X 50 μL SYBR Green (1 : 10,000) (Invitrogen) for 30 min in the dark. Cell suspensions were washed three times with PBS and analysed by flow cytometry (FACS-Canto II, Becton Dickinson). FlowJo 7.5 (Tree Star, Inc.) was used for analysing the data [51]. The assay was made by pooling all the sera from each group due to the low serum volume available.

Local and Systemic Tolerance: Repeat Doses in Mice.
There were no deaths in any of the groups of animals when evaluating local toxicity due to repeat doses of the IMPIPS mixture. No local adverse reactions were observed (postimmunisation), such as erythema, oedema, eschar, or necrosis, at the inoculation site in any of the groups.  Figure 1). Statistical analysis revealed no significant differences between the groups (p > 0:05).
The temperature of male mice immunised with PSS, IMPIPS+adjuvant, and IMPIPS+PSS ranged from 30.72°C to 32.27°C, 29.56°C to 32.63°C, and 30.57°C to 32.23°C, respectively, during the study, whilst for females immunised with PSS, IMPIPS+adjuvant, and IMPIPS+PSS, it ranged from 31.05°C to 37.57°C, 29.62°C to 33.91°C, and 31.2°C to 33.35°C, respectively (Supplementary Figure 1). Overall, temperatures remained between the minimum and maximum ranges of the control group.

Haematological Parameters.
The haematic picture evaluated parameters related to erythrocytes (erythrocyte count (RBC), haematocrit (HCT), haemoglobin (Hb), and total plasma proteins (TPP)) and leukocytes (leukocyte (WBC) count) for determining possible alterations caused by the formulation. The reference values for analysing each biochemical parameter were determined by mean control ± SD; no significant differences (p > 0:05) were observed regarding either erythrocytes or leukocytes. Complete blood cell count (CBC) values came within the stated parameters, except for day 3 when control group females had a slight reduction in haematocrit and haemoglobin, possibly due to previous bleedings. By contrast, an increase in leukocytes was observed in control group males; this increase could have been caused by stress due to the bleeding.
Renal function was evaluated by measuring BUN and CRE. ANOVA analysis identified no statistically significant differences between the different treatments when comparing values between the groups or when comparing control group values (saline solution) to those for the other study groups (p > 0:05) (Figure 2). The reference values for analysing each biochemical parameter were determined from the means for the controls ± SD (Figure 2).

Histopathology.
Microcirculatory changes related to slight and moderate vascular congestion were observed in the myocardium in 2/4 females immunised with the IMPIPS+adjuvant formulation; there was no evidence of congestion in the other animals from the same group or from the other groups (p > 0:05). No microcirculatory changes were seen in any of the immunised animals, such as oedema and/or haemorrhage, inflammatory infiltrate, structural changes, or binucleation ( Figure 3).
Slight congestion was observed in the kidneys of at least one animal from every group. These changes occurred more in females than in males, since a lesion was found in just one male compared to 6 females (2 from each group) in which congestion was observed. No microcirculatory changes such as oedema and haemorrhage, inflammatory infiltrate, structural changes, or binucleations were observed in any of the study groups (Figure 4).
No macroscopic or microscopic alterations were observed when analysing the duodenum, though 50% of the mice immunised with IMPIPS+adjuvant had mixed inflammatory infiltrate in the mesentery ( Figure 5). Likewise, follicular hyperplasia was observed in 100% of the mice immunised with IMPIPS+adjuvant when analysing lymphoid tissue, 50% being slight and 50% moderate. 37.5% of the mice immunised with IMPIPS+PSS had this reaction to a slight degree and 25% to a moderate degree. Two (33.3%) control group mice had slight follicular hyperplasia ( Figure 6). A scale was generated to semiquantify follicular hyperplasia according to the number of nodules using a magnification of 400x (Supplementary Table 2).

Immunogenicity
3.3.1. Determining Anti-IMPIPS Serum Ability to Recognise P. falciparum-Infected RBC by IFA. An indirect immunofluorescence assay was used for determining anti-IMPIPS serum ability to recognise pRBC. The serum from mice immunised with IMPIPS+adjuvant as well as that from those immunised with IMPIPS+PSS was able to recognise pRBC ( Figure 7). Considering that some P. berghei proteins share high identity with their P. falciparum counterparts, such as enolase [55], the higher fluorescence intensity observed in the positive control might be due to the higher number of proteins being recognised versus just the six blood-stage proteins being recognised from animals immunised with the peptide mixture.

Determining Anti-IMPIPS Antibodies' Merozoite
Invasion Inhibition Capability. The functional role of antibodies stimulated by immunisation with IMPIPS was determined by an in vitro invasion inhibition assay. The serum from animals immunised with the IMPIPS peptide mix-ture+PSS was able to inhibit invasion, maximum values being 61.84% for males and 68.34% for females. Likewise, the serum from the animals immunised with the IMPIPS peptide mixture+adjuvant had 67.62% maximum invasion inhibition values for males and 70.82% for females. It was found that inhibition was concentration dependent (p < 0:05). No statistical difference was observed between inhibition percentages for the females compared to those for the males (p > 0:05) ( Figure 8). Sera surpassing 70% were considered strong inhibitors, whilst those ranging from 50% to 69% were considered medium-high inhibitors. Those having 30% to 49% were considered medium-low inhibitors, those from 10%-29% are low inhibitors, and those < 9% were considered negative.

Discussion
Toxicological studies of the formulation to be used in clinical studies are of the utmost importance when developing vaccines as they provide information about possible adverse effects which might arise due to the formulation, either at the inoculation site or in the different organs and tissues of subjects being vaccinated [37,38]. This study thus evaluated the safety and immunogenicity of a mixture of 23 modified peptides derived from 8 Spz proteins (CSP-1, TRAP, STARP, SPECT-1 and SPECT-2, CelTOS, and SIAP-1 and SIAP-2) and 6 Mrz proteins (AMA-1, EBA-175, EBA-140, SERA-5, MSP-1, and HRP-II) [9][10][11] in a murine model as a synthetic antimalarial vaccine candidate. The single dose local tolerance study was aimed at evaluating the site exposed to the formulation 72 h postimmunisation; no adverse reactions such as erythema, oedema, eschar, or necrosis were observed in the mice immunised with the IMPIPS mixture+PSS or in those immunised with IMPIPS +adjuvant. This suggested that the IMPIPS mixture did not produce local toxic effects due to single dose SC immunisation. The forgoing led to continuing local and systemic tolerance studies regarding repeat doses (4 immunisations) where SC immunisation also did not produce adverse effects such as erythema, oedema, eschar, or necrosis at the administration site, suggesting that repeat IMPIPS doses did not produce irritation or toxicity at the immunisation site.
Male and female mice immunised with the IMPIPS peptide mixture+PSS or IMPIPS+adjuvant gained weight and increased their weekly food consumption, and their body temperature was within established parameters (i.e., regarding the formulation's systemic effects) [56], thereby supporting the idea that the IMPIPS mixture did not affect physiology.
Blood chemistry analysis showed that creatinine values did not exceed the parameters compared to control values  during the first bleeding. Creatinine values on day 40 exceeded the parameters; however, they became reduced by day 70, coming within normal parameters for the males. Such transitory increase could have been due to stress or dehydration since, unlike other mammals, mice excrete creatinine in their urine. Once the situation had become resolved, creatinine returned to its normal values [56]. BUN values came within normal parameters. Since no other damage was observed on day 70, the histological study of the kidneys verified that an increase in creatinine was due to prerenal causes and not to renal damage.
Regarding histological analysis, the microcirculatory changes in the myocardium compatible with congestion did not arise from administering the IMPIPS peptide mixture, since control group animals also had this pathology. Such changes could mainly have been due to the hypovolemic shock caused by the final bleeding; this would have occurred because haemorrhagic shock affects tissue perfusion [57].
Mixed inflammatory infiltrate was observed in the histological study of the mesentery; this description refers to localised accumulations of mononuclear and polymorphonuclear cells, indicating the presence of a foreign body in acute phase, i.e., causing the antigenic stimulus to continue. This finding in the animals immunised with IMPIPS+adjuvant and not in those immunised with IMPIPS+PPS or in the control group indicated that the antigen continued being active. This could have been caused by the formation of a deposit at the injection site due to the adjuvant's mechanism of action (doses 2, 3, and 4 of the formulation were administered by IP route) [58].
The nodular hyperplasia observed in animals' lymphoid tissue is mainly due to normal lymphoid nodule inflammation in response to an antigen. Such response in this case was triggered by the immunisation; such reaction is also known as reactive lymphoid hyperplasia [59] which, as expected, was much stronger in the animals immunised with the formulation containing IMPIPS+adjuvant.
FIDIC's previous studies have shown that individual immunisation of IMPIPS in Aotus monkeys has stimulated the production of antibodies which have been able to recognise parasite proteins in their native form and induce a protective immune response, determined by the total absence of parasites in the blood following experimental challenge [30,32,33]. The present study highlighted the fact that serum from male and female mice immunised with the IMPIPS mixture+adjuvant or IMPIPS+PSS recognised the parasite in the Mrz stage. This indicated that although the peptides had been modified for their presentation by human MHC-II [27,29,31], the mixture was capable of inducing an immune response against the native proteins from which they were derived, even in a murine model, thereby reinforcing the idea of using IMPIPS in an antimalarial vaccine [60]. Such response was seen in the immunofluorescence and invasion inhibition assays.

Conclusions
Local tolerance and systemic safety tests regarding single and repeat doses in this study showed no toxicity induced by the IMPIPS mixture in a murine model 70 days after the first immunisation, reaffirming that peptide-based vaccines can represent a safe option. The IMPIPS mixture was immunogenic in a murine model, even when the peptides were designed for human MHC-II. Such results suggested that the IMPIPS mixture is safe and thus further immunogenicity and protection assays in a nonhuman primate model such as the Aotus spp. monkey but delivered with adjuvants authorised for human use are recommended.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.