Systems Analysis of Protective Immune Responses to RTS,S Malaria Vaccination in Humans
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Systems analysis of protective immune responses to RTS,S malaria vaccination in humans Dmitri Kazmina,1, Helder I. Nakayab,1, Eva K. Leec, Matthew J. Johnsond, Robbert van der Moste, Robert A. van den Bergf, W. Ripley Ballouf, Erik Jongerte, Ulrike Wille-Reeceg, Christian Ockenhouseg, Alan Aderemh, Daniel E. Zakh, Jerald Sadoffi, Jenny Hendriksi, Jens Wrammerta, Rafi Ahmeda,2, and Bali Pulendrana,j,2 aEmory Vaccine Center, Emory University, Atlanta, GA 30329; bSchool of Pharmaceutical Sciences, University of Sao Paulo, São Paulo 05508, Brazil; cSchool of Industrial and Systems Engineering, Georgia Institute of Technology, Atlanta, GA 30332; dCenter for Genome Engineering, University of Minnesota, Minneapolis, MN 55108; eGSK Vaccines, Rixensart 1330, Belgium; fGSK Vaccines, Rockville, MD 20850; gProgram for Appropriate Technology in Health-Malaria Vaccine Initiative, Washington, DC 20001; hCenter for Infectious Disease Research, Seattle, WA 98109; iCrucell, Leiden 2333, The Netherlands; and jDepartment of Pathology, Emory University School of Medicine, Atlanta, GA 30322 Contributed by Rafi Ahmed, January 4, 2017 (sent for review December 19, 2016; reviewed by Elias Haddad and Robert Seder) RTS,S is an advanced malaria vaccine candidate and confers trials conducted in malaria endemic areas in Africa proved the significant protection against Plasmodium falciparum infection in vaccine to be partially protective in adults (9), children (10, 11), humans. Little is known about the molecular mechanisms driving and infants (12, 13). These results were further confirmed in a vaccine immunity. Here, we applied a systems biology approach phase III trial in sub-Saharan Africa (14–17) in which 55.8% to study immune responses in subjects receiving three consecutive efficacy against clinical malaria was observed over the first 12 mo immunizations with RTS,S (RRR), or in those receiving two immuni- of follow-up in children of 5–17 mo (14). zations of RTS,S/AS01 following a primary immunization with ade- The magnitude of the CSP-specific antibody responses induced novirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. by RTS,S/AS01 vaccination has been correlated with protection in Subsequent controlled human malaria challenge (CHMI) of the vac- previous studies (18). However, RTS,S/AS01 vaccination does not + + cinees with Plasmodium-infected mosquitoes, 3 wk after the final induce CD8 T cells, and because CD8 T cells have a critical role immunization, resulted in ∼50% protection in both groups of vac- in protection against malaria (19), this observation provided one cinees. Circumsporozoite protein (CSP)-specific antibody titers, pre- rationale to include a viral vector in a prime-boost regimen with challenge, were associated with protection in the RRR group. In RTS,S/AS01 to determine whether this addition enhances antibody, SYSTEMS BIOLOGY contrast, ARR-induced lower antibody responses, and protection + + + CD4 ,andCD8 T-cell responses, which synergize to confer enhanced was associated with polyfunctional CD4 T-cell responses 2 wk after protection against infection. In this context, replication-defective priming with Ad35. Molecular signatures of B and plasma cells de- recombinant adenoviral vectors (rAds) are known to potently induce tected in PBMCs were highly correlated with antibody titers prechal- T-cell immunity and are lead vaccine candidates (20). Thus, to lenge and protection in the RRR cohort. In contrast, early signatures augment cellular responses to the RTS,S /AS01 vaccine, a com- of innate immunity and dendritic cell activation were highly associ- bination of adenoviral vaccine candidates and RTS,S/AS01 has ated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and Significance predicted protection. These results suggest that protective immu- nity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecu- The RTS,S malaria vaccine is the most advanced malaria vaccine lar correlates of protection to vaccination. candidate to be tested in humans. Despite its promise, there is little understanding of its mechanism of action. In this work, malaria | vaccine | systems vaccinology | systems biology | immune we describe the use of a systems biological approach to iden- tify “molecular signatures” that are induced rapidly after the standard RTS,S vaccination regimen, consisting of three RTS,S alaria is a communicable disease transmitted by mosqui- Anopheles. immunizations, or with a different regimen consisting of a Mtoes from the genus There was an estimated primary immunization with recombinant adenovirus 35 (Ad35) 214 million cases of malaria in 2014, with an estimated 438,000 expressing the circumsporozoite malaria antigen followed by deaths, primarily in sub-Saharan Africa. Nearly three-quarters of two immunizations with RTS,S. These results reveal important malaria victims were children younger than 5, with an estimated insights about the innate and adaptive responses to vaccina- 800 childhood deaths daily (1). tion and identify signatures of protective immunity against A malarial vaccine candidate targeting circumsporozoite pro- malaria. tein (CSP), a major component of the Plasmodium falciparum sporozoite coat, has been developed and recommended for pilot Author contributions: D.K., R.v.d.M., R.A.v.d.B., W.R.B., E.J., U.W.-R., C.O., A.A., J.S., J.H., implementation by the World Health Organization (2). The vac- R.A., and B.P. designed research; D.K., H.I.N., E.K.L., M.J.J., and J.W. performed research; J.S. and J.H. contributed new reagents/analytic tools; D.K., H.I.N., A.A., D.E.Z., R.A., and cine candidate, named RTS,S/AS01, consists of 19 NANP repeats B.P. analyzed data; R.v.d.M. and R.A.v.d.B. contributed to the development of the clinical (R) and the C-terminal of CSP including T-cell epitopes (T) fused study; R.A.v.d.B. contributed to the analysis plan; and D.K., H.I.N., and B.P. wrote to hepatitis B surface antigen (HBsAg) (S) (3, 4). It is produced as the paper. a mixture of the fusion construct (RTS) with native HBsAg (S), Reviewers: E.H., Drexel University; and R.S., National Institutes of Health. which self-assembles into virus-like particles with the CSP portion Conflict of interest statement: R.v.d.M., R.A.v.d.B., W.R.B., and E.J. are employees of the of the fusion protein exposed on the surface. The RTS,S/AS01 GSK group of companies. They report ownership of GSK shares and/or restricted GSK shares. vaccine candidate contains adjuvant system AS01, a liposome- O ′ Data deposition: The data reported in this paper have been deposited in the Gene Ex- based adjuvant comprising 3- -desacyl-4 -monophosphoryl lipid pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE89292). A (MPL), a Toll-like receptor 4 ligand, and QS-21, a saponin 1D.K. and H.I.N. contributed equally to this work. extracted from the bark of the Quillaja saponaria Molina tree (5). 2To whom correspondence may be addressed. Email: [email protected] or rahmed@ To date, RTS,S/AS01 has been shown to have an acceptable emory.edu. safety and immunogenicity profile in controlled human malaria This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. infection (CHMI) and field (6–8) studies. Phase IIa/IIb clinical 1073/pnas.1621489114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1621489114 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 also been evaluated (21). Recently, an Ad35-CSP (AdVac)–RTS, immunizations. This was surprising because RRR vaccination S/AS01 prime-boost approach was tested in humans, and its ef- induced a greater magnitude of CSP-antibody titers compared ficacy and immunogenicity was compared with the RTS,S/AS01 with ARR vaccination (Fig. S1A). This discordance may reflect vaccine candidate alone (18). Surprisingly, however, inclusion of differences in kinetics of the ASC response induced by ARR the adenoviral prime immunization did not result in increased versus RRR. Alternatively, a different population of ASC (which vaccine efficacy (18). was not sampled in this study) may contribute to enhanced an- In this study, we sought to enhance our understanding of the tibody response in the RRR group. + + mechanisms of vaccine-induced protection against malaria. In re- CSP-specific CD4 and CD8 T-cell responses to vaccination + cent years, the tools of systems biology (22, 23) have been applied to were also assessed (18). There was negligible induction of CD8 identify signatures of immunogenicity to vaccination and have T-cell responses by RRR and a modest induction by ARR. In con- + provided insights into the mechanisms of immune responses in- trast, there was a significant induction of CSP-specific CD4 T-cell duced by vaccines such as the live attenuated yellow fever (YF-17D) response by ARR, and to a much weaker degree by RRR (18). and seasonal influenza vaccines (24–26). Here, we used systems The functionality of T cells was monitored by FACS analysis using approaches to trace the temporal variations of the transcriptional a panel including four markers: CD40L, IL-2, TNFα,andIFNγ.In response elicited by the two vaccines and to identify transcriptional the ARR vaccine group, there was a markedly enhanced frequency signatures associated with protection and immunogenicity. of polyfunctional (expressing three or four functions) CSP-specific + CD4 T cells at D14, D42, and D77, and postchallenge (Fig. S1D). Results Challenge Model for the RTS,S/AS01 and AdVac Malaria Vaccines. The Immunologic Correlates of Protection. As reported (18), in the clinical trial (NCT01366534) was conducted at Walter Reed Army RRR vaccine arm, individuals who did not develop parasitemia Institute of Research, as described (18). Forty-six healthy malaria- within 28 d after challenge (referred hereafter as “protected”) naïve volunteers, randomized to two study arms, participated in this had higher concentration of anti-CSP antibodies at the time of study testing the efficacy of RTS,S and AdVac malaria vaccine challenge than nonprotected individuals (Fig.