HIV-1 neutralizing antibodies in SHIV-infected macaques recapitulate structurally divergent modes of human V2 apex recognition with a single D gene.

Broadly neutralizing antibodies targeting the V2 apex of the HIV-1 envelope trimer are among the most common specificities elicited in HIV-1-infected humans and simian-human immunodeficiency virus (SHIV)-infected macaques. To gain insight into the prevalent induction of these antibodies, we isolated and characterized 11 V2 apex-directed neutralizing antibody lineages from SHIV-infected rhesus macaques. Remarkably, all SHIV-induced V2 apex lineages were derived from reading frame two of the rhesus DH3-15*01 gene. Cryo-EM structures of envelope trimers in complex with antibodies from nine rhesus lineages revealed modes of recognition that mimicked three canonical human V2 apex-recognition modes. Notably, amino acids encoded by DH3-15*01 played divergent structural roles, inserting into a hole at the trimer apex, H-bonding to an exposed strand, or forming part of a loop scaffold. Overall, we identify a DH3-15*01-signature for rhesus V2 apex broadly neutralizing antibodies and show that highly selected genetic elements can play multiple roles in antigen recognition. Highlights: Isolated 11 V2 apex-targeted HIV-neutralizing lineages from 10 SHIV-infected Indian-origin rhesus macaquesCryo-EM structures of Fab-Env complexes for nine rhesus lineages reveal modes of recognition that mimic three modes of human V2 apex antibody recognitionAll SHIV-elicited V2 apex lineages, including two others previously published, derive from the same DH3-15*01 gene utilizing reading frame twoThe DH3-15*01 gene in reading frame two provides a necessary, but not sufficient, signature for V2 apex-directed broadly neutralizing antibodiesStructural roles played by DH3-15*01-encoded amino acids differed substantially in different lineages, even for those with the same recognition modePropose that the anionic, aromatic, and extended character of DH3-15*01 in reading frame two provides a selective advantage for V2 apex recognition compared to B cells derived from other D genes in the naïve rhesus repertoireDemonstrate that highly selected genetic elements can play multiple roles in antigen recognition, providing a structural means to enhance recognition diversity.

Assessing the impact of autologous neutralizing antibodies on rebound dynamics in postnatally SHIV-infected ART-treated infant rhesus macaques.

The presence of antibodies against HIV in infected children is associated with a greater capacity to control viremia in the absence of therapy. While the benefits of early antiretroviral treatment (ART) in infants are well documented, early ART may interfere with the development of antibody responses. In contrast to adults, early treated children lack detectable HIV-specific antibodies, suggesting a fundamental difference in HIV pathogenesis. Despite this potential adverse effect, early ART may decrease the size of the latent reservoir established early in infection in infants, which can be beneficial in viral control. Understanding the virologic and immunologic aspects of pediatric HIV is crucial to inform innovative targeted strategies for treating children living with HIV. In this study, we investigate how ART initiation time sets the stage for trade-offs in the latent reservoir establishment and the development of humoral immunity and how these, in turn, affect posttreatment dynamics. We also elucidate the biological function of antibodies in pediatric HIV. We employ mathematical modeling coupled with experimental data from an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT four weeks after birth and started treatment at different times after infection. In addition to viral load measurements, antibody responses and latent reservoir sizes were measured. We estimate model parameters by fitting viral load measurements to the standard HIV viral dynamics model within a nonlinear fixed effects framework. This approach allows us to capture differences between rhesus macaques (RMs) that develop antibody responses or exhibit high latent reservoir sizes compared to those that do not. We find that neutralizing antibody responses are associated with increased viral clearance and decreased viral infectivity but decreased death rate of infected cells. In addition, the presence of detectable latent reservoir is associated with less robust immune responses. These results demonstrate that both immune response and latent reservoir dynamics are needed to understand post-rebound dynamics and point to the necessity of a comprehensive approach in tailoring personalized medical interventions.

Comparative analysis of within-host dynamics of acute infection and viral rebound dynamics in postnatally SHIV-infected ART-treated infant rhesus macaques.

Viral dynamics of acute HIV infection and HIV rebound following suspension of antiretroviral therapy may be qualitatively similar but must differ given, for one, development of adaptive immune responses. Understanding the differences of acute HIV infection and viral rebound dynamics in pediatric populations may provide insights into the mechanisms of viral control with potential implications for vaccine design and the development of effective targeted therapeutics for infants and children. Mathematical models have been a crucial tool to elucidate the complex processes driving viral infections within the host. Traditionally, acute HIV infection has been modeled with a standard model of viral dynamics initially developed to explore viral decay during treatment, while viral rebound has necessitated extensions of that standard model to incorporate explicit immune responses. Previous efforts to fit these models to viral load data have underscored differences between the two infection stages, such as increased viral clearance rate and increased death rate of infected cells during rebound. However, these findings have been predicated on viral load measurements from disparate adult individuals. In this study, we aim to bridge this gap, in infants, by comparing the dynamics of acute infection and viral rebound within the same individuals by leveraging an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Ten infant Rhesus macaques (RMs) orally challenged with SHIV.C.CH505 375H dCT and given ART at 8 weeks post-infection. These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. We use the HIV standard viral dynamics model fitted to viral load measurements in a nonlinear mixed effects framework. We find that the primary difference between acute infection and rebound is the increased death rate of infected cells during rebound. We use these findings to generate hypotheses on the effects of adaptive immune responses. We leverage these findings to formulate hypotheses to elucidate the observed results and provide arguments to support the notion that delayed viral rebound is characterized by a stronger CD8+ T cell response.

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection.

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development.

Antibodies targeting the fusion peptide on the HIV envelope provide protection to rhesus macaques against mucosal SHIV challenge.

The fusion peptide (FP) on the HIV-1 envelope (Env) trimer can be targeted by broadly neutralizing antibodies (bNAbs). Here, we evaluated the ability of a human FP-directed bNAb, VRC34.01, along with two vaccine-elicited anti-FP rhesus macaque mAbs, DFPH-a.15 and DF1W-a.01, to protect against simian-HIV (SHIV)BG505 challenge. VRC34.01 neutralized SHIVBG505 with a 50% inhibitory concentration (IC50) of 0.58 µg/ml, whereas DF1W-a.01 and DFPH-a.15 were 4- or 30-fold less potent, respectively. VRC34.01 was infused into four rhesus macaques at a dose of 10 mg/kg and four rhesus macaques at a dose of 2.5 mg/kg. The animals were intrarectally challenged 5 days later with SHIVBG505. In comparison with all 12 control animals that became infected, all four animals infused with VRC34.01 (10 mg/kg) and three out of four animals infused with VRC34.01 (2.5 mg/kg) remained uninfected. Because of the lower potency of DF1W-a.01 and DFPH-a.15 against SHIVBG505, we infused both Abs at a higher dose of 100 mg/kg into four rhesus macaques each, followed by SHIVBG505 challenge 5 days later. Three of four animals that received DF1W-a.01 were protected against infection, whereas all animals that received DFPH-a.15 were protected. Overall, the protective serum neutralization titers observed in these animals were similar to what has been observed for other bNAbs in similar SHIV infection models and in human clinical trials. In conclusion, FP-directed mAbs can thus provide dose-dependent in vivo protection against mucosal SHIV challenges, supporting the development of prophylactic vaccines targeting the HIV-1 Env FP.

Host immunity associated with spontaneous suppression of viremia in therapy-naïve young rhesus macaques following neonatal SHIV infection.

IMPORTANCE: Despite the advent of highly active anti-retroviral therapy, people are still dying from HIV-related causes, many of whom are children, and a protective vaccine or cure is needed to end the HIV pandemic. Understanding the nature and activation states of immune cell subsets during infection will provide insights into the immunologic milieu associated with viremia suppression that can be harnessed via therapeutic strategies to achieve a functional cure, but these are understudied in pediatric subjects. We evaluated humoral and adaptive host immunity associated with suppression of viremia in rhesus macaques infected soon after birth with a pathogenic SHIV. The results from our study provide insights into the immune cell subsets and functions associated with viremia control in young macaques that may translate to pediatric subjects for the design of future anti-viral strategies in HIV-1-infected infants and children and contribute to an understudied area of HIV-1 pathogenesis in pediatric subjects.

Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

Assessing the impact of autologous neutralizing antibodies on viral rebound in postnatally SHIV-infected ART-treated infant rhesus macaques.

While the benefits of early antiretroviral therapy (ART) initiation in perinatally infected infants are well documented, early ART initiation is not always possible in postnatal pediatric HIV infections, which account for the majority of pediatric HIV cases worldwide. The timing of onset of ART initiation is likely to affect the size of the latent viral reservoir established, as well as the development of adaptive immune responses, such as the generation of neutralizing antibody responses against the virus. How these parameters impact the ability of infants to control viremia and the time to viral rebound after ART interruption is unclear. To gain insight into the dynamics, we utilized mathematical models to investigate the effect of time of ART initiation via latent reservoir size and autologous virus neutralizing antibody responses in delaying viral rebound when treatment is interrupted. We used an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model that mimics breast milk HIV transmission in human infants. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT and either given ART at 4-7 days post-infection (early ART condition), at 2 weeks post-infection (intermediate ART condition), or at 8 weeks post-infection (late ART condition). These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. We develop a stochastic mathematical model to investigate the joint effect of latent reservoir size, the autologous neutralizing antibody potency, and CD4+ T cell levels on the time to viral rebound and control of post-rebound viral loads. We find that the latent reservoir size is an important determinant in explaining time to viral rebound by affecting the growth rate of the virus. The presence of neutralizing antibodies also can delay rebound, but we find this effect for high potency antibody responses only.

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques.

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. SHIV.C.CH505 is an extensively characterized virus encoding the TF HIV-1 Env CH505 mutated at position 375 shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides Δ375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure.

Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates.

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells' recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells' predominant CD8+ effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses.

Neonatal SHIV infection in rhesus macaques elicited heterologous HIV-1-neutralizing antibodies.

Infants and children infected with human immunodeficiency virus (HIV)-1 have been shown to develop neutralizing antibodies (nAbs) against heterologous HIV-1 strains, characteristic of broadly nAbs (bnAbs). Thus, having a neonatal model for the induction of heterologous HIV-1 nAbs may provide insights into the mechanisms of neonatal bnAb development. Here, we describe a neonatal model for heterologous HIV-1 nAb induction in pathogenic simian-HIV (SHIV)-infected rhesus macaques (RMs). Viral envelope (env) evolution showed mutations at multiple sites, including nAb epitopes. All 13 RMs generated plasma autologous HIV-1 nAbs. However, 8/13 (62%) RMs generated heterologous HIV-1 nAbs with increasing potency over time, albeit with limited breadth, and mapped to multiple nAb epitopes, suggestive of a polyclonal response. Moreover, plasma heterologous HIV-1 nAb development was associated with antigen-specific, lymph-node-derived germinal center activity. We define a neonatal model for heterologous HIV-1 nAb induction that may inform future pediatric HIV-1 vaccines for bnAb induction in infants and children.

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies.

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency. IMPORTANCE An effective HIV-1 vaccination strategy will need to stimulate rare precursor B cells of multiple bNAb lineages and affinity mature them along desired pathways. Here, we searched for V2-apex germ line-targeting Envs among a large set of diverse primate lentiviruses and identified minimally modified versions of one chimpanzee SIV Env that bound several human V2-apex bNAb precursors and stimulated one of these in a V2-apex bNAb precursor-expressing knock-in mouse. We also generated chimeric simian-chimpanzee immunodeficiency viruses and showed that they elicit low-titer V2-directed heterologous plasma breadth in six of seven infected rhesus macaques. Characterization of this antibody response identified a new class of weakly cross-reactive neutralizing antibodies that target the V2-apex, but only in occluded-open Env trimers. The existence of this cryptic epitope, which in some Env backgrounds is immunodominant, needs to be considered in immunogen design.

Strategies for HIV-1 vaccines that induce broadly neutralizing antibodies.

After nearly four decades of research, a safe and effective HIV-1 vaccine remains elusive. There are many reasons why the development of a potent and durable HIV-1 vaccine is challenging, including the extraordinary genetic diversity of HIV-1 and its complex mechanisms of immune evasion. HIV-1 envelope glycoproteins are poorly recognized by the immune system, which means that potent broadly neutralizing antibodies (bnAbs) are only infrequently induced in the setting of HIV-1 infection or through vaccination. Thus, the biology of HIV-1-host interactions necessitates novel strategies for vaccine development to be designed to activate and expand rare bnAb-producing B cell lineages and to select for the acquisition of critical improbable bnAb mutations. Here we discuss strategies for the induction of potent and broad HIV-1 bnAbs and outline the steps that may be necessary for ultimate success.

Stabilized HIV-1 envelope immunization induces neutralizing antibodies to the CD4bs and protects macaques against mucosal infection.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4+ T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine-induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

Leveraging antigenic seniority for maternal vaccination to prevent mother-to-child transmission of HIV-1.

The development of a maternal HIV vaccine to synergize with current antiretroviral drug prophylaxis can overcome implementation challenges and further reduce mother-to-child transmission (MTCT) of HIV. Both the epitope-specificity and autologous neutralization capacity of maternal HIV envelope (Env)-specific antibodies have been implicated in decreased risk of MTCT of HIV. Our goal was to determine if heterologous HIV Env immunization of SHIV.C.CH505-infected, ART-suppressed female rhesus macaques (RMs) could boost autologous Env-specific antibodies. SHIV.C.CH505-infected female RMs (n = 12), began a daily ART regimen at 12 weeks post-infection (wpi), which was continued for 12 weeks. Starting 2 weeks after ART initiation, RMs received 3 monthly immunizations with HIV b.63521/1086.C gp120 or placebo (n = 6/group) vaccine with adjuvant STR8S-C. Compared to the placebo-immunized animals, Env-vaccinated, SHIV-infected RMs exhibited enhanced IgG binding, avidity, and ADCC responses against the vaccine immunogens and the autologous SHIV.C.CH505 Env. Notably, the Env-specific memory B cells elicited by heterologous vaccination were dominated by cells that recognized the SHIV.C.CH505 Env, the antigen of primary exposure. Thus, vaccination of SHIV-infected, ART-suppressed RMs with heterologous HIV Envs can augment multiple components of the antibody response against the Env antigen of primary exposure, suggesting antigenic seniority. Our results suggest that a universal maternal HIV vaccination regimen can be developed to leverage antigenic seniority in targeting the maternal autologous virus pool.

Repeated exposure to heterologous hepatitis C viruses associates with enhanced neutralizing antibody breadth and potency.

A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically related, antibody-sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody-sensitive viruses is a promising approach to inducing potent bNAbs in humans.

Zoonotic origin of the human malaria parasite Plasmodium malariae from African apes.

The human parasite Plasmodium malariae has relatives infecting African apes (Plasmodium rodhaini) and New World monkeys (Plasmodium brasilianum), but its origins remain unknown. Using a novel approach to characterise P. malariae-related sequences in wild and captive African apes, we found that this group comprises three distinct lineages, one of which represents a previously unknown, highly divergent species infecting chimpanzees, bonobos and gorillas across central Africa. A second ape-derived lineage is much more closely related to the third, human-infective lineage P. malariae, but exhibits little evidence of genetic exchange with it, and so likely represents a separate species. Moreover, the levels and nature of genetic polymorphisms in P. malariae indicate that it resulted from the zoonotic transmission of an African ape parasite, reminiscent of the origin of P. falciparum. In contrast, P. brasilianum falls within the radiation of human P. malariae, and thus reflects a recent anthroponosis.