Richard E. Stafford

Protection Against Malaria by Intravenous Immunization with a Nonreplicating Sporozoite Vaccine

Malaria Sporozoite Vaccine Each year, hundreds of millions of people are infected with Plasmodium falciparum, the mosquito-borne parasite that causes malaria. A preventative vaccine is greatly needed. Seder et al. (p. 1359, published online 8 August; see the Perspective by Good) now report the results from a phase I clinical trial where subjects were immunized intravenously with a whole, attenuated sporozoite vaccine. Three of 9 subjects who received four doses and zero of 6 subjects who received five doses of the vaccine went on to develop malaria after controlled malaria infection. Both antibody titers and cellular immune responses correlated positively with the dose of vaccine received, suggesting that both arms of the adaptive immune response may have participated in the observed protection. Intravenous immunization with an attenuated whole malaria sporozoite vaccine protected volunteers in a phase I clinical trial. [Also see Perspective by Good] Consistent, high-level, vaccine-induced protection against human malaria has only been achieved by inoculation of Plasmodium falciparum (Pf) sporozoites (SPZ) by mosquito bites. We report that the PfSPZ Vaccine—composed of attenuated, aseptic, purified, cryopreserved PfSPZ—was safe and wel-tolerated when administered four to six times intravenously (IV) to 40 adults. Zero of six subjects receiving five doses and three of nine subjects receiving four doses of 1.35 × 105 PfSPZ Vaccine and five of six nonvaccinated controls developed malaria after controlled human malaria infection (P = 0.015 in the five-dose group and P = 0.028 for overall, both versus controls). PfSPZ-specific antibody and T cell responses were dose-dependent. These data indicate that there is a dose-dependent immunological threshold for establishing high-level protection against malaria that can be achieved with IV administration of a vaccine that is safe and meets regulatory standards.

Live Attenuated Malaria Vaccine Designed to Protect through Hepatic CD8+ T Cell Immunity

The efficacy of a sporozoite-based malaria vaccine is tested in humans, nonhuman primates, and mice. Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8+ T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8+, IFN-γ–producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria

Immunization of volunteers by the bite of mosquitoes carrying radiation-attenuated Plasmodium falciparum sporozoites protects greater than 90% of such volunteers against malaria, if adequate numbers of immunizing biting sessions and sporozoite-infected mosquitoes are used. Nonetheless, until recently it was considered impossible to develop, license and commercialize a live, whole parasite P. falciparum sporozoite (PfSPZ) vaccine. In 2003 Sanaria scientists reappraised the potential impact of a metabolically active, non-replicating PfSPZ vaccine, and outlined the challenges to producing such a vaccine. Six years later, significant progress has been made in overcoming these challenges. This progress has enabled the manufacture and release of multiple clinical lots of a 1(st) generation metabolically active, non-replicating PfSPZ vaccine, the Sanaria PfSPZ Vaccine, submission of a successful Investigational New Drug application to the US Food and Drug Administration, and initiation of safety, immunogenicity and protective efficacy studies in volunteers in MD, US. Efforts are now focused on how best to achieve submission of a successful Biologics License Application and introduce the vaccine to the primary target population of African children in the shortest possible period of time. This will require implementation of a systematic, efficient clinical development plan. Short term challenges include optimizing the (1) efficiency and scale up of the manufacturing process and quality control assays, (2) dosage regimen and method of administration, (3) potency of the vaccine, and (4) logistics of delivering the vaccine to those who need it most, and finalizing the methods for vaccine stabilization and attenuation. A medium term goal is to design and build a facility for manufacturing highly potent and stable vaccine for pivotal Phase 3 studies and commercial launch.

Protection against malaria at 1 year and immune correlates following PfSPZ vaccination

An attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine, PfSPZ Vaccine, is highly protective against controlled human malaria infection (CHMI) 3 weeks after immunization, but the durability of protection is unknown. We assessed how vaccine dosage, regimen, and route of administration affected durable protection in malaria-naive adults. After four intravenous immunizations with 2.7 × 105 PfSPZ, 6/11 (55%) vaccinated subjects remained without parasitemia following CHMI 21 weeks after immunization. Five non-parasitemic subjects from this dosage group underwent repeat CHMI at 59 weeks, and none developed parasitemia. Although Pf-specific serum antibody levels correlated with protection up to 21–25 weeks after immunization, antibody levels waned substantially by 59 weeks. Pf-specific T cell responses also declined in blood by 59 weeks. To determine whether T cell responses in blood reflected responses in liver, we vaccinated nonhuman primates with PfSPZ Vaccine. Pf-specific interferon-γ-producing CD8 T cells were present at ∼100-fold higher frequencies in liver than in blood. Our findings suggest that PfSPZ Vaccine conferred durable protection to malaria through long-lived tissue-resident T cells and that administration of higher doses may further enhance protection.

Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.

Attenuated PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection

Significance A highly effective malaria vaccine capable of long-term protection against genetically diverse strains is urgently needed. Here, we demonstrate that a three-dose regimen of a live attenuated whole-parasite malaria vaccine conferred durable sterile protection through 33 weeks in ∼50% of subjects against a controlled human malaria infection strain that is heterologous to the vaccine strain. Prior studies by others and us have shown that T cells are critical to mediating sterile protection after live-attenuated malaria vaccination. Here, we provide evidence that this Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine) induces antigen-specific IFN-γ-producing CD8 and CD4 T cells that recognize both the homologous and the heterologous Pf strain. A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 105 PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35–87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls (P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36–99%) remained without parasitemia compared with none of six nonvaccinated controls. PfSPZ-specific T-cell and antibody responses were detected in all vaccine recipients. Cytokine production by T cells from vaccinated subjects after in vitro stimulation with homologous (NF54) or heterologous (7G8) PfSPZ were highly correlated. Interestingly, PfSPZ-specific T-cell responses in the blood peaked after the first immunization and were not enhanced by subsequent immunizations. Collectively, these data suggest durable protection against homologous and heterologous Pf parasites can be achieved with PfSPZ Vaccine. Ongoing studies will determine whether protective efficacy can be enhanced by additional alterations in the vaccine dose and number of immunizations.

Corrigendum: Protection against malaria at 1 year and immune correlates following PfSPZ vaccination

Nat. Med.; 10.1038/nm.4110; corrected online 18 May 2016 In the version of this article initially published online, the authors omitted a funding source, The Bill and Melinda Gates Foundation (Investment ID: 24922). The error has been corrected for the print, PDF and HTML versions of this article.

Protection against inhalation anthrax by immunization with Salmonella enterica serovar Typhi Ty21a stably producing protective antigen of Bacillus anthracis

The national blueprint for biodefense concluded that the United States is underprepared for biological threats. The licensed anthrax vaccine absorbed vaccine, BioThrax, requires administration of at least 3–5 intramuscular doses. The anthrax vaccine absorbed vaccine consists of complex cell-free culture filtrates of a toxigenic Bacillus anthracis strain and causes tenderness at the injection site and significant adverse events. We integrated a codon-optimized, protective antigen gene of B. anthracis (plus extracellular secretion machinery), into the chromosome of the licensed, oral, live-attenuated typhoid fever vaccineTy21a to form Ty21a-PA-01 and demonstrated excellent expression of the gene encoding protective antigen. We produced the vaccine in a 10-L fermenter; foam-dried and vialed it, and characterized the dried product. The vaccine retained ~50% viability for 20 months at ambient temperature. Sera from animals immunized by the intraperitoneal route had high levels of anti-protective antigen antibodies by enzyme-linked immunosorbent assay and anthrax lethal toxin-neutralizing activity. Immunized mice were fully protected against intranasal challenge with ~5 LD50 of B. anthracis Sterne spores, and 70% (7/10) of vaccinated rabbits were protected against aerosol challenge with 200 LD50 of B. anthracis Ames spores. There was a significant correlation between protection and antibody levels determined by enzyme-linked immunosorbent assay and toxin-neutralizing activity. These data provide the foundation for achievement of our ultimate goal, which is to develop an oral anthrax vaccine that is stable at ambient temperatures and induces the rapid onset of durable, high-level protection after a 1-week immunization regimen.Counterterrorism: creating a safe, stable, easily stored anthrax vaccineA vaccine candidate for anthrax infection shows promise for improving preparedness for a biological attack. Bacillus anthracis, the bacterium responsible for anthrax is a top-tier bioterrorism agent due to its high lethality and spore stability. The current FDA-approved anthrax vaccine and other vaccine candidates in development lack ease of preparation, have short shelf lives and adverse effects. B. Kim Lee Sim of Protein Potential LLC and her collaborators combined key B. anthracis genetic material into an existing typhoid vaccine. The vaccine vector possesses high stability, a strong safety record, and offers long-term protection after oral administration, which Sim’s group hopes to preserve in their candidate anthrax vaccine. The team showed that their hybrid vaccine conferred excellent protection in rabbits and a short vaccination regimen, and suggest further studies into its suitability for human vaccine studies.

Erratum: Protection against malaria at 1 year and immune correlates following PfSPZ vaccination (Nature Medicine (2016) DOI: 10.1038/nm.4110))

Nat. Med.; 10.1038/nm.4110; corrected online 18 May 2016 In the version of this article initially published online, the authors omitted a funding source, The Bill and Melinda Gates Foundation (Investment ID: 24922). The error has been corrected for the print, PDF and HTML versions of this article.

Erratum: Corrigendum: Protection against malaria at 1 year and immune correlates following PfSPZ vaccination

Nat. Med.; 10.1038/nm.4110; corrected online 18 May 2016 In the version of this article initially published online, the authors omitted a funding source, The Bill and Melinda Gates Foundation (Investment ID: 24922). The error has been corrected for the print, PDF and HTML versions of this article.