HIV vaccines induce CD8+ T cells with low antigen receptor sensitivity.

Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.

Superior antibody immunogenicity of a viral-vectored RH5 blood-stage malaria vaccine in Tanzanian infants as compared to adults.

BACKGROUND: RH5 is a leading blood-stage candidate antigen for a Plasmodium falciparum vaccine; however, its safety and immunogenicity in malaria-endemic populations are unknown. METHODS: A phase 1b, single-center, dose-escalation, age-de-escalation, double-blind, randomized, controlled trial was conducted in Bagamoyo, Tanzania (NCT03435874). Between 12th April and 25th October 2018, 63 healthy adults (18-35 years), young children (1-6 years), and infants (6-11 months) received a priming dose of viral-vectored ChAd63 RH5 or rabies control vaccine. Sixty participants were boosted with modified vaccinia virus Ankara (MVA) RH5 or rabies control vaccine 8 weeks later and completed 6 months of follow-up post priming. Primary outcomes were the number of solicited and unsolicited adverse events post vaccination and the number of serious adverse events over the study period. Secondary outcomes included measures of the anti-RH5 immune response. FINDINGS: Vaccinations were well tolerated, with profiles comparable across groups. No serious adverse events were reported. Vaccination induced RH5-specific cellular and humoral responses. Higher anti-RH5 serum immunoglobulin G (IgG) responses were observed post boost in young children and infants compared to adults. Vaccine-induced antibodies showed growth inhibition activity (GIA) in vitro against P. falciparum blood-stage parasites; their highest levels were observed in infants. CONCLUSIONS: The ChAd63-MVA RH5 vaccine shows acceptable safety and reactogenicity and encouraging immunogenicity in children and infants residing in a malaria-endemic area. The levels of functional GIA observed in RH5-vaccinated infants are the highest reported to date following human vaccination. These data support onward clinical development of RH5-based blood-stage vaccines to protect against clinical malaria in young African infants. FUNDING: Medical Research Council, London, UK.

Cytotoxicity of human antibodies targeting the circumsporozoite protein is amplified by 3D substrate and correlates with protection.

Human monoclonal antibodies (hmAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on the sporozoite surface are a promising tool for preventing malaria infection. However, their mechanisms of protection remain unclear. Here, using 13 distinctive PfCSP hmAbs, we provide a comprehensive view of how PfCSP hmAbs neutralize sporozoites in host tissues. Sporozoites are most vulnerable to hmAb-mediated neutralization in the skin. However, rare but potent hmAbs additionally neutralize sporozoites in the blood and liver. Efficient protection in tissues mainly associates with high-affinity and high-cytotoxicity hmAbs inducing rapid parasite loss-of-fitness in the absence of complement and host cells in vitro. A 3D-substrate assay greatly enhances hmAb cytotoxicity and mimics the skin-dependent protection, indicating that the physical stress imposed on motile sporozoites by the skin is crucial for unfolding the protective potential of hmAbs. This functional 3D cytotoxicity assay can thus be useful for downselecting potent anti-PfCSP hmAbs and vaccines.

Cryo-EM structures of anti-malarial antibody L9 with circumsporozoite protein reveal trimeric L9 association and complete 27-residue epitope.

Monoclonal antibody L9 recognizes the Plasmodium falciparum circumsporozoite protein (PfCSP) and is highly protective following controlled human malaria challenge. To gain insight into its function, we determined cryoelectron microscopy (cryo-EM) structures of L9 in complex with full-length PfCSP and assessed how this recognition influenced protection by wild-type and mutant L9s. Cryo-EM reconstructions at 3.6- and 3.7-Å resolution revealed L9 to recognize PfCSP as an atypical trimer. Each of the three L9s in the trimer directly recognized an Asn-Pro-Asn-Val (NPNV) tetrapeptide on PfCSP and interacted homotypically to facilitate L9-trimer assembly. We analyzed peptides containing different repeat tetrapeptides for binding to wild-type and mutant L9s to delineate epitope and homotypic components of L9 recognition; we found both components necessary for potent malaria protection. Last, we found the 27-residue stretch recognized by L9 to be highly conserved in P. falciparum isolates, suggesting the newly revealed complete L9 epitope to be an attractive vaccine target.

Low-Dose Subcutaneous or Intravenous Monoclonal Antibody to Prevent Malaria.

BACKGROUND: New approaches for the prevention and elimination of malaria, a leading cause of illness and death among infants and young children globally, are needed. METHODS: We conducted a phase 1 clinical trial to assess the safety and pharmacokinetics of L9LS, a next-generation antimalarial monoclonal antibody, and its protective efficacy against controlled human malaria infection in healthy adults who had never had malaria or received a vaccine for malaria. The participants received L9LS either intravenously or subcutaneously at a dose of 1 mg, 5 mg, or 20 mg per kilogram of body weight. Within 2 to 6 weeks after the administration of L9LS, both the participants who received L9LS and the control participants underwent controlled human malaria infection in which they were exposed to mosquitoes carrying Plasmodium falciparum (3D7 strain). RESULTS: No safety concerns were identified. L9LS had an estimated half-life of 56 days, and it had dose linearity, with the highest mean (±SD) maximum serum concentration (Cmax) of 914.2±146.5 µg per milliliter observed in participants who had received 20 mg per kilogram intravenously and the lowest mean Cmax of 41.5±4.7 µg per milliliter observed in those who had received 1 mg per kilogram intravenously; the mean Cmax was 164.8±31.1 in the participants who had received 5 mg per kilogram intravenously and 68.9±22.3 in those who had received 5 mg per kilogram subcutaneously. A total of 17 L9LS recipients and 6 control participants underwent controlled human malaria infection. Of the 17 participants who received a single dose of L9LS, 15 (88%) were protected after controlled human malaria infection. Parasitemia did not develop in any of the participants who received 5 or 20 mg per kilogram of intravenous L9LS. Parasitemia developed in 1 of 5 participants who received 1 mg per kilogram intravenously, 1 of 5 participants who received 5 mg per kilogram subcutaneously, and all 6 control participants through 21 days after the controlled human malaria infection. Protection conferred by L9LS was seen at serum concentrations as low as 9.2 µg per milliliter. CONCLUSIONS: In this small trial, L9LS administered intravenously or subcutaneously protected recipients against malaria after controlled infection, without evident safety concerns. (Funded by the National Institute of Allergy and Infectious Diseases; VRC 614 ClinicalTrials.gov number, NCT05019729.).

The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria.

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 ß-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs.

Protective effects of combining monoclonal antibodies and vaccines against the Plasmodium falciparum circumsporozoite protein.

Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human "repeat" mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.

The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo.

Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP central repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and central repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria.

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope-CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

Antigenic Restimulation of Virus-Specific Memory CD8+ T Cells Requires Days of Lytic Protein Accumulation for Maximal Cytotoxic Capacity.

In various infections or vaccinations of mice or humans, reports of the persistence and the requirements for restimulation of the cytotoxic mediators granzyme B (GrB) and perforin (PRF) in CD8+ T cells have yielded disparate results. In this study, we examined the kinetics of PRF and GrB mRNA and protein expression after stimulation and associated changes in cytotoxic capacity in virus-specific memory cells in detail. In patients with controlled HIV or cleared respiratory syncytial virus (RSV) or influenza virus infections, all virus-specific CD8+ T cells expressed low PRF levels without restimulation. Following stimulation, they displayed similarly delayed kinetics for lytic protein expression, with significant increases occurring by days 1 to 3 before peaking on days 4 to 6. These increases were strongly correlated with, but were not dependent upon, proliferation. Incremental changes in PRF and GrB percent expression and mean fluorescence intensity (MFI) were highly correlated with increases in HIV-specific cytotoxicity. mRNA levels in HIV-specific CD8+ T-cells exhibited delayed kinetics after stimulation as with protein expression, peaking on day 5. In contrast to GrB, PRF mRNA transcripts were little changed over 5 days of stimulation (94-fold versus 2.8-fold, respectively), consistent with posttranscriptional regulation. Changes in expression of some microRNAs, including miR-17, miR-150, and miR-155, suggested that microRNAs might play a significant role in regulation of PRF expression. Therefore, under conditions of extremely low or absent antigen levels, memory virus-specific CD8+ T cells require prolonged stimulation over days to achieve maximal lytic protein expression and cytotoxic capacity.IMPORTANCE Antigen-specific CD8+ T cells play a major role in controlling most virus infections, primarily by perforin (PRF)- and granzyme B (GrB)-mediated apoptosis. There is considerable controversy regarding whether PRF is constitutively expressed, rapidly increased similarly to a cytokine, or delayed in its expression with more prolonged stimulation in virus-specific memory CD8+ T cells. In this study, the degree of cytotoxic capacity of virus-specific memory CD8+ T cells was directly proportional to the content of lytic molecules, which required antigenic stimulation over several days for maximal levels. This appeared to be modulated by increases in GrB transcription and microRNA-mediated posttranscriptional regulation of PRF expression. Clarifying the requirements for maximal cytotoxic capacity is critical to understanding how viral clearance might be mediated by memory cells and what functions should be induced by vaccines and immunotherapies.

Dual RNA-seq identifies human mucosal immunity protein Mucin-13 as a hallmark of Plasmodium exoerythrocytic infection.

The exoerythrocytic stage of Plasmodium infection is a critical window for prophylactic intervention. Using genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we show that the human mucosal immunity gene, mucin-13 (MUC13), is strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm MUC13 transcript increases in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, marking both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes. This data provides insights into host-parasite interactions in Plasmodium infection, and demonstrates that a component of host mucosal immunity is reprogrammed during the progression of infection.

Private sector drug shops frequently dispense parenteral anti-malarials in a rural region of Western Uganda.

BACKGROUND: Malaria is a leading cause of paediatric morbidity and mortality in Uganda. More than half of febrile children in rural areas initially seek care at private clinics and drug shops. These shops are generally unregulated and the quality of clinical care is variable, with the potential for misdiagnosis and the development of drug resistance. There is thus an urgent need to identify rural drug shops and coordinate their malaria treatment efforts with those of the public sector. The objective of the study was to identify all drug shops in the Bugoye sub-county of Western Uganda and assess their anti-malarial dispensing practices. METHODS: This study is a cross-sectional survey of drug shops in a rural sub-county of Western Uganda. In the first phase, shop locations, licensing and shopkeeper's qualifications, and supply and pricing of anti-malarials were characterized. In the second phase, the proportion of anti-malarials dispensed by private drug shops was compared to public health facilities. RESULTS: A total of 48 drug shops were identified. Only one drug shop (1 of 48, 2%) was licensed with the sub-county's records office. The drug shops stocked a variety of anti-malarials, including first-line therapies and less effective agents (e.g., sulfadoxine/pyrimethamine). Almost all drug shops (45 of 48, 94%) provided parenteral anti-malarials. Of the 3900 individuals who received anti-malarials during the study, 2080 (53.3%) purchased anti-malarials through the private sector compared to 1820 (46.7%) who obtained anti-malarials through the public sector. Drug shops were the primary source of parenteral anti-malarials. Inadequate dosing of anti-malarials was more common in drug shops. CONCLUSIONS: Drug shops are major sources of parenteral anti-malarials, which should be reserved for cases of severe malaria. Strengthening malaria case management and incorporating drug shops in future interventions is necessary to optimize malaria control efforts in the sub-county, and in similarly endemic regions.

A possible role for dupilumab (Dupixent) in the management of idiopathic chronic eczematous eruption of aging.

Aging individuals can develop generalized, exquisitely-pruritic, eczematous eruptions of uncertain etiology that can be therapeutically-refractory and life-altering. Limited information exists in the literature to guide clinicians in the diagnosis and management of such patients. It is suggested that in approximately 40% of such patients a known cause for their chronic pruritic eruptions cannot be identified. In this report we will refer to this subgroup of patients as having idiopathic chronic eczematous eruption of aging (CEEA). Idiopathic CEEA must be distinguished from other established eczematous dermatoses. Idiopathic CEEA patients often require long-term systemic immunosuppressive drugs to make living bearable. Elder-onset atopic dermatitis is the most difficult of the known dermatoses to distinguish from idiopathic CEEA. Because of their clinical similarities we questioned whether dupilumab (Dupixent®), the first FDA-approved biologic for atopic dermatitis, might be valuable in the management of idiopathic CEEA. We report the case of an elderly man with idiopathic CEEA of four-years' duration who had a complete clinical response to the initiation of treatment with dupilumab. This case is presented to stimulate more discussion and systematic study of a possible role for dupilumab in otherwise-refractory idiopathic CEEA patients. We also propose a set of diagnostic criteria for idiopathic CEEA.

Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors.

Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers that are associated with diarrhea. The genome of EAEC strain 042, a diarrheal pathogen validated in a human challenge study, encodes multiple colonization factors. Notable among them are aggregative adherence fimbriae (AAF/II) and a secreted antiaggregation protein (Aap). Deletion of aap is known to increase adherence, autoaggregation, and biofilm formation, so it was proposed that Aap counteracts AAF/II-mediated interactions. We hypothesized that Aap sterically masks heat-resistant agglutinin 1 (Hra1), an integral outer membrane protein recently identified as an accessory colonization factor. We propose that this masking accounts for reduced in vivo colonization upon hra1 deletion and yet no colonization-associated phenotypes when hra1 is deleted in vitro. Using single and double mutants of hra1, aap, and the AAF/II structural protein gene aafA, we demonstrated that increased adherence in aap mutants occurs even when AAF/II proteins are genetically or chemically removed. Deletion of hra1 together with aap abolishes the hyperadherence phenotype, demonstrating that Aap indeed masks Hra1. The presence of all three colonization factors, however, is necessary for optimal colonization and for rapidly building stacked-brick patterns on slides and cultured monolayers, the signature EAEC phenotype. Altogether, our data demonstrate that Aap serves to mask nonstructural adhesins such as Hra1 and that optimal colonization by EAEC is mediated through interactions among multiple surface factors. IMPORTANCE Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers of the human intestine and can cause diarrhea. Compared to other E. coli pathogens, little is known about the genes and pathogenic mechanisms that differentiate EAEC from harmless commensal E. coli. EAEC bacteria attach via multiple proteins and structures, including long appendages produced by assembling molecules of AafA and a short surface protein called Hra1. EAEC also secretes an antiadherence protein (Aap; also known as dispersin) which remains loosely attached to the cell surface. This report shows that dispersin covers Hra1 such that the adhesive properties of EAEC seen in the laboratory are largely produced by AafA structures. When the bacteria colonize worms, dispersin is sloughed off, or otherwise removed, such that Hra1-mediated adherence occurs. All three factors are required for optimal colonization, as well as to produce the signature EAEC stacked-brick adherence pattern. Interplay among multiple colonization factors may be an essential feature of exceptional colonizers.

Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics.

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.