Role of human Pegivirus infections in whole Plasmodium falciparum sporozoite vaccination and controlled human malaria infection in African volunteers

Abstract

Background: Diverse vaccination outcomes and protection levels among different populations pose a serious challenge to the development of an effective malaria vaccine. Co-infections are among many factors associated with immune dysfunction and sub-optimal vaccination outcomes. Chronic, asymptomatic viral infections can contribute to the modulation of vaccine efficacy through various mechanisms. Human Pegivirus-1 (HPgV-1) persists in immune cells thereby potentially modulating immune responses. We investigated whether Pegivirus infection influences vaccine-induced responses and protection in African volunteers undergoing whole P. falciparum sporozoites-based malaria vaccination and controlled human malaria infections (CHMI).

Methods: HPgV-1 prevalence was quantified by RT-qPCR in plasma samples of 96 individuals before, post vaccination with PfSPZ Vaccine and after CHMI in cohorts from Tanzania and Equatorial Guinea. The impact of HPgV-1 infection was evaluated on (1) systemic cytokine and chemokine levels measured by Luminex, (2) PfCSP-specific antibody titers quantified by ELISA, (3) asexual blood-stage parasitemia pre-patent periods and parasite multiplication rates, (4) HPgV-1 RNA levels upon asexual blood-stage parasitemia induced by CHMI.

Results: The prevalence of HPgV-1 was 29.2% (28/96) and sequence analysis of the 5' UTR and E2 regions revealed the predominance of genotypes 1, 2 and 5. HPgV-1 infection was associated with elevated systemic levels of IL-2 and IL-17A. Comparable vaccine-induced anti-PfCSP antibody titers, asexual blood-stage multiplication rates and pre-patent periods were observed in HPgV-1 positive and negative individuals. However, a tendency for higher protection levels was detected in the HPgV-1 positive group (62.5%) compared to the negative one (51.6%) following CHMI. HPgV-1 viremia levels were not significantly altered after CHMI.

Conclusions: HPgV-1 infection did not alter PfSPZ Vaccine elicited levels of PfCSP-specific antibody responses and parasite multiplication rates. Ongoing HPgV-1 infection appears to improve to some degree protection against CHMI in PfSPZ-vaccinated individuals. This is likely through modulation of immune system activation and systemic cytokines as higher levels of IL-2 and IL17A were observed in HPgV-1 infected individuals. CHMI is safe and well tolerated in HPgV-1 infected individuals. Identification of cell types and mechanisms of both silent and productive infection in individuals will help to unravel the biology of this widely present but largely under-researched virus.

Keywords: Antibody response; Controlled human malaria infection; Human pegivirus; Immune activation; Malaria; PfSPZ vaccine.

Fig. 1

Unbiased search for RNA molecules encoding human viruses in RNA-seq transcriptomics data. a Overall prevalence of 9 human viruses detected in 172 whole blood samples b Number of viral RNA-seq reads detected for each of the identified viruses. Virus names are plotted on the y-axis and proportion (a), number of reads (B) on the x-axis. c Distribution of the 9 different viruses across the 28 individuals included. Virus names are plotted on the y-axis and volunteer IDs on the x-axis. Each bar indicates viral reads for an individual. The log viral RNA-seq reads are plotted, in increasing order ranging from 0 to 3; green indicating low number and red high number of reads

Fig. 2

Proportion of individuals with (purple) and without (grey) HPgV- 1 infection. a Total cohort of 96 vacinees, b separated by gender, c Country of origin, d HIV-1 infection status. All individuals are between 18 and 35 years of age. Chi square with Yates correction for group comparisons (*P < 0.05)

Fig. 3

Comparisons of HPgV-1 viral loads. No differences in HPgV-1 viral loads between Equatorial Guinea (green, n = 16) and Tanzania (blue, n = 12) volunteers (a). Two distinct groups with low (blue) and high (grey) viremia levels in plasma are found in HPgV-1 infected individuals (b). The two groups were divided based on a cut off value of 600,000 RNA copies/ml plasma

Fig. 4

Phylogenetic inferences of the HPgV-1 isolates based on 5′ UTR. Phylogenetic tree was constructed using Neighbour joining method and Kimura two-parameter model of the 5′ UTR. The 5′ UTR sequences from Tanzania and Equatorial Guinea (n = 26) were compared to selected references spanning genotype 1 to 7 from different countries available in the NCBI database. The accession numbers for the reference sequences were: AF488786, AF488789, KC618399, KP710602, U36388, JX494177, Y16436, and MF398547 (Genotype 1, Pink); AB003289, AF104403, D90600, JX494179, MG229668, JX494180, U4402, U59518 (Genotype 2; 2a light brown), MH000566, U59529, U63715, MH053130 (Genotype 2; 2b Brown); AB008335, KR108695, JX494176, D87714 (Genotype 3, Green); AB0188667, AB021287, HQ3311721 (Genotype 4, Maroon); DQ117844, AY949771, AF488796, AF488797 (Genotype 5, Light blue), AB003292, AF177619 (Genotype 6, Bright green); HQ331235, HQ 3312233 (Genotype 7, Golden) and Hepatitis C (AJ132997, Black) was used as outgroup

Fig. 5

Phylogenetic inferences of the HPgV-1 isolates based on E2 region. Phylogenetic tree was constructed using Neighbour joining method and Kimura two-parameter model of the E2 region of HPgV-1. The E2 sequences from Tanzania and Equatorial Guinea (n = 9) were compared to selected references spanning genotype 1 to 6 from different countries available in the NCBI database including; KP701602.1, KM670109, U36380, KP710600, KC618399, AB003291 (Genotype 1, Pink); AF121950, MK686596, D90600 (Genotype 2; 2a Brown), U63715 (Genotype 2; 2b Brown) D87714 (Genotype 3, Green); AB0188667 (Genotype 4, Brown); AY94977, KC618401, AY951979 (Genotype 5, Light blue) and AB003292 (Genotype 6, Green). Equatorial Guinean and Tanzanian strains identified in this study are denoted by strain number followed with letters EG or TZ, respectively (Red). Chimpanzee HPgV-1 strain (AF70476, Black) was used as outgroup and U4402 (Genotype 2, Golden) was used for mapping of our sequences to identify regions of similarity. The scale bar under the tree indicates nucleotide substitution per site

Fig. 6

HPgV-1 infection is associated with increased systemic levels of IL-2 and IL-17A. Cytokine, chemokine and growth factors levels were analysed by Luminex and levels compared between HPgV-1 negative (5′ UTR-, grey, n = 35) and HPgV-1 positive (5′ UTR + , purple, n = 9) volunteers. Absolute serum concentrations levels (pg/ml) of Interluekin-2 (IL-2) and Interluekin-17A (IL-17A) at samples taken before vaccination are shown. Significantly higher IL-2 and IL-17A are seen in the HPgV-1⁺ compared to the HPgV-1⁻. Wilcoxon rank sum test was used to determine significance (P value * < 0.05) which are indicated on top of top for each group comparison

Fig. 7

HPgV-1 infection status does not impact on anti-PfCSP antibody titres. Total IgG antibodies recognizing full length PfCSP were measured by ELISA in HPgV-1 negative (5′ UTR-, grey), HPgV-1 positive (5′ UTR + , purple) volunteers. a Shows baseline (pre-vaccination) anti-PfCSP IgG levels of HPgV-1 negative (n = 47) compared to HPgV-1 positive (n = 23) volunteers. b Anti-PfCSP IgG levels at 14 days past last vaccination in HPgV-1 positive individuals (n = 17) versus the HPgV-1 negative (n = 37) group. c, d Comparison of vaccine-induced changes in anti-PfCSP IgG titres as net responses (14 days post last immunization—baseline) as well as fold (14 days post last vaccination/baseline). Only vaccinated individuals were included for 14 days post last immunization, net and fold change responses. One HPgV-1⁺ individual was not included in these subsequent analyses due to missing antibody data. Log anti-PfCSP titres expressed in arbitrary units are shown. Each point represent an individual, box plot with horizontal bar show median values for each group. Statistical significance was calculated by using Wilcoxon rank sum test (P value * < 0.05). P values are indicated on top for each group comparison

Fig. 8

HPgV-1 infection does not influence P. falciparum pre-patent periods and parasite multiplication rates during CHMI. Parasitemia was determined in whole blood by qPCR and thick blood smear microscopy (TBS). The analysis included only placebo participants, positive and negative for HPgV-1. a Shows log-fold change of parasitemia in 48 h between HPgV-1 negative (5′ UTR-, grey, n = 13) and HPgV-1 positive (5′ UTR + , purple, n = 7) volunteers. b Comparison of days post CHMI to malaria positivity by microscopy in HPgV-1 negative (5′ UTR-, grey, n = 11) and HPgV-1 positive (5′ UTR + , purple, n = 7). c HPgV-1 viral loads before (red) and 28 days post CHMI (green) in HPgV-1 infected individuals. Each point represents an individual, box plots show data distribution with horizontal bar denoting viral load at each visit. Lines connect viremia levels in individuals found positive for HPgV-1 on both time points. Geometric means were compared between groups and unpaired t-test was used to calculate significance. Horizontal bars represent mean with standard deviation Wilcoxon rank sum test was used to compare viremia levels before and after CHMI. P-values are indicated on top of each comparison

Fig. 9

Association of HPgV-1 infection status with PfSPZ CHMI outcome and anti-CSP titers in immunized volunteers. Individuals were treated with either normal saline (placebo) or PfSPZ Vaccine (vaccinees). Presence or absence of malaria parasites was determined in whole blood by thick blood smear microscopy (TBS) and confirmed by qPCR. Total IgG antibodies recognizing full length PfCSP were measured by ELISA. a Proportion of non-protected (cream) and protected (blue) in vaccinated volunteers with and without HPgV-1 infection. Proportions are indicated inside the bar and volunteer numbers on top. c Total anti-CSP IgG levels at 14 days past last vaccination in the protected (malaria negative) and non-protected (malaria positive) groups, with and without HPgV-1 infection. Log anti-PfCSP titres expressed in O.D units are shown. Each point represent an individual, and box plot with horizontal bar show median values for each group. Chi square with Yates correction was used for group comparisons of categorical values (*P < 0.05). Wilcoxon rank sum test was used to compare anti-CSP titres in the two groups. P values are indicated on top of each comparison