Further observations on hydrogen peroxide antisepsis and COVID-19 cases among healthcare workers and inpatients.

BACKGROUND: The use of prophylactic antisepsis to protect against coronavirus disease 2019 (COVID-19) has been suggested. This study investigated hydrogen peroxide antisepsis (HPA) at two hospitals in Ghana. METHODS: Cases of COVID-19 among healthcare workers (HCWs) using hydrogen peroxide (HP-HCWs) or not using hydrogen peroxide (NHP-HCWs), vaccinated or unvaccinated, were recorded at Shai-Osudoku Hospital (SODH), Dodowa, and Mount Olives Hospital (MOH), Techiman, between May 2020 and December 2021. The effect of HPA in all inpatients at MOH was also observed. Permutation tests were used to determine P values. FINDINGS: At SODH, there were 62 (13.5%) cases of COVID-19 among 458 NHP-HCWs but no cases among eight HP-HCWs (P=0.622) from May to December 2020. Between January and March 2021, 10 (2.7%) of 372 NHP-HCWs had COVID-19, but there were no cases among 94 HP-HCWs (P=0.206). At MOH, prior to HPA, 17 (20.2%) of 84 HCWs and five (1.4%) of 370 inpatients had COVID-19 in July 2020. From August 2020 to March 2021, two of 54 (3.7%) HCWs who stopped HPA had COVID-19; none of 32 NHP-HCWs contracted COVID-19. At SODH, none of 23 unvaccinated HP-HCWs and 35 (64%) of 55 unvaccinated NHP-HCWs had COVID-19 from April to December 2021 (P<0.0001). None of 34 vaccinated HP-HCWs and 53 (13.6%) of 390 vaccinated NHP-HCWs had COVID-19 (P=0.015). No inpatients on prophylactic HPA (total 7736) contracted COVID-19. CONCLUSION: Regular, daily HPA protects HCWs from COVID-19, and curtails nosocomial spread of SARS-CoV-2.

Restricted or absent immune responses in human populations to Plasmodium falciparum gamete antigens that are targets of malaria transmission-blocking antibodies.

We have studied the antibodies to sexual stage antigens of Plasmodium falciparum in human sera from Papua New Guinea where intense transmission of P. falciparum occurs as well as the less prevalent P. malariae and P. vivax. In extracts of gametes of P. falciparum we have studied the reactivity of serum antibodies with antigens labeled with 125I on the surface of the gametes as well as intracellular gamete antigens. A prominent 27-kD sexual stage-specific intracellular protein was recognized more or less in proportion to the general antibody response to gamete proteins. The response to the gamete surface proteins, however, was quite unrepresentative of the general antibody response to the intracellular gamete proteins. No antibodies were detected against Pfs25, a 21-kD protein expressed on zygotes and ookinetes of P. falciparum and known to be a sensitive target of malaria transmission-blocking antibodies. The antibody response to two other target antigens of transmission-blocking antibodies on the surface of gametes of P. falciparum, a 230- and a 48- and 45-kD protein doublet, was very variable and independent of the response to the internal protein antigens. Several possibilities are discussed that may account for the variable response to these gamete surface antigens in individuals with otherwise good antibody responses to internal sexual stage proteins. Among these is the possibility that there is MHC restriction of the immune response to the gamete surface antigens in the human population. This interpretation accords well with evidence for MHC-restricted immune response to the same P. falciparum gamete surface antigens in studies with H-2 congenic mice (24).

Allelic forms of gp195, a major blood-stage antigen of Plasmodium falciparum, are expressed in liver stages.

Mature exoerythrocytic (EE) forms of two cloned lines (3D7 and HB3) of Plasmodium falciparum were obtained in the livers of splenectomized chimpanzees. Sectioned preparations were examined by immunofluorescence (IFA) using mAbs that distinguished allelic variants of the blood-form antigen gp195 and mAbs that recognized multiple conserved epitopes of gp195. EE forms and blood schizonts exhibited identical IFA reactions for each respective clone, showing that the antigen was expressed identically in liver and blood-stage parasites. A third chimpanzee was infected with sporozoites derived from a mixture of 3D7 and HB3 gametocytes that had undergone cross-fertilization in the mosquitoes. IFAs on the EE forms in this animal showed that segregation of each gp195 allele had occurred earlier in the life cycle, providing evidence that the parasite is haploid for the whole of its mammalian development.

Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals.

Pfs25 is a sexual stage antigen of Plasmodium falciparum that is expressed on the surface of zygote and ookinete forms of the parasite. Monoclonal antibodies directed against native Pfs25 can block completely the development of P. falciparum oocysts in the midgut of the mosquito vector. Thus, this 25-kD protein is a potential vaccine candidate for eliciting transmission-blocking immunity in inhabitants of malaria endemic regions. We have synthesized, by secretion from yeast, a polypeptide analogue of Pfs25 that reacts with conformation-dependent monoclonal antibodies, and elicits transmission-blocking antibodies when used to immunize mice and monkeys in conjunction with a muramyl tripeptide adjuvant. Our results suggest the further evaluation of recombinant DNA-derived Pfs25 in transmission-blocking vaccination studies in humans.

Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant vaccinia virus.

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal antibodies that block transmission of malaria from vertebrate host to mosquito vector. The surface of mammalian cells infected with a recombinant vaccinia virus that expressed Pfs25 specifically bound transmission-blocking monoclonal antibodies. Furthermore, major histocompatibility complex-disparate congenic mouse strains immunized with recombinant Pfs25 elicited transmission-blocking antibodies, demonstrating that the capacity to develop transmission-blocking antibodies is not genetically restricted in mice. Live recombinant viruses may provide an inexpensive, easily administered alternative to subunit vaccines prepared from purified recombinant proteins to block transmission of malaria in developing countries.

Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria.

Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.

DNA cloning of Plasmodium falciparum circumsporozoite gene: amino acid sequence of repetitive epitope.

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS beta-lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

Limited immunological recognition of critical malaria vaccine candidate antigens.

Current vaccine development strategies for malaria depend on widespread immunological responsiveness to candidate antigens such as the zygote surface antigens and the sporozoite coat protein, the circumsporozoite (CS) protein. Since immunological responsiveness is controlled mainly by genes mapping within the major histocompatibility complex (MHC), the humoral immune response to the zygote surface antigens and the cytotoxic T lymphocyte (CTL) response to the CS protein were examined in MHC-disparate congenic mouse strains. Only two of six strains responded to the 230-kilodalton zygote surface antigen and another two strains responded to the 48/45-kilodalton surface antigen. From two mouse strains, expressing between them five different class I MHC molecules, there was recognition of only a single CTL epitope from the CS protein, which was from a polymorphic segment of the molecule. The restricted CTL response to this protein parallels the restricted antibody response to this protein observed in humans and mice. These findings suggest that subunit malaria vaccines now being developed may be ineffective.

Genetic analysis of the human malaria parasite Plasmodium falciparum.

Malaria parasites are haploid for most of their life cycle, with zygote formation and meiosis occurring during the mosquito phase of development. The parasites can be analyzed genetically by transmitting mixtures of cloned parasites through mosquitoes to permit cross-fertilization of gametes to occur. A cross was made between two clones of Plasmodium falciparum differing in enzymes, drug sensitivity, antigens, and chromosome patterns. Parasites showing recombination between the parent clone markers were detected at a high frequency. Novel forms of certain chromosomes, detected by pulsed-field gradient gel electrophoresis, were produced readily, showing that extensive rearrangements occur in the parasite genome after cross-fertilization. Since patients are frequently infected with mixtures of genetically distinct parasites, mosquito transmission is likely to provide the principal mechanisms for generating parasites with novel genotypes.

An antimalarial neem leaf extract has both schizonticidal and gametocytocidal activities.

A crude acetone/water (50/50) extract of neem leaves (IRAB) was evaluated for activity against the asexual (trophozoites/schizonts) and the sexual (gametocytes) forms of the malarial parasite, Plasmodium falciparum, in vitro. In separate 72 hour cultures of both asexual parasites and mature gametocytes treated with IRAB (0.5 microg/mL), parasite numbers were less than 50% of the numbers in control cultures, which had 8.0% and 8.5% parasitemia, respectively. In cultures containing 2.5 microg/mL, asexual parasites and mature and immature gametocytes were reduced to 0.1%, 0.2%, and 0% parasitemia, respectively. There were no parasites in the cultures containing 5.0 microg/mL. This extract, if found safe, may provide materials for development of new antimalarial drugs that may be useful both in treatment of malaria as well as the control of its transmission through gametocytes.

Conserved and variant epitopes of target antigens of transmission-blocking antibodies among isolates of Plasmodium falciparum from Malaysia.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.

Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum.

Malaria was transmitted to six normal human volunteers by mosquitoes infected from cultured gametocytes of Plasmodium falciparum. This method, which offers advantages over other methods of infecting volunteers, will be useful for evaluating the efficacy of human malaria vaccines.

Differential non-responsiveness in humans of candidate Plasmodium falciparum vaccine antigens.

Synthetic subunit vaccines to sporozoites, merozoites, and gametes are being developed for malaria. The vaccine strategy assumes that the population to be immunized will respond favorably to these vaccine antigens. Using sera of 35 adults and 50 children from the The Gambia, West Africa, where Plasmodium falciparum is highly endemic, we examined the humoral immune response to candidate malaria vaccine antigens from sporozoites, merozoites, and gametes. We observed widespread restricted immunogenicity to defined parasite antigens in children and adults. HLA typing of adult lymphocytes demonstrated a marked diversity in HLA haplotypes in this population. Our results and those from our studies in mice suggest that genetic factors may partly explain the immunological non-responsiveness. This may necessitate re-evaluation of the malaria vaccine strategy.

The epidemiology of Plasmodium falciparum malaria in two Cameroonian villages: Simbok and Etoa.

In support of ongoing immunologic studies on immunity to Plasmodium falciparum, demographic, entomologic, parasitologic, and clinical studies were conducted in two Cameroonian villages located 3 km apart. Simbok (population = 907) has pools of water present year round that provide breeding sites for Anopheles gambiae, whereas Etoa (population = 485) has swampy areas that dry up annually in which A. funestus breed. Results showed that individuals in Simbok receive an estimated 1.9 and 1.2 infectious bites per night in the wet and dry season, respectively, whereas individuals in Etoa receive 2.4 and 0.4 infectious bites per night, respectively. Although transmission patterns differ, the rate of acquisition of immunity to malaria appears to be similar in both villages. A prevalence of 50-75% was found in children < 10 years old, variable levels in children 11-15 years old, and 31% in adults. Thus, as reported in other parts of Africa, individuals exposed to continuous transmission of P. falciparum slowly acquired significant, but not complete, immunity.

Identification of anti-Plasmodium falciparum antibodies in human breast milk.

Malarial infections are rarely observed in neonates. It has been postulated that some immunity may be passively transferred during nursing, but anti-malarial antibodies (Abs) have not been detected in human milk. In this study, milk samples, collected 2-14 days after parturition from women at the Central Maternity Hospital, Yaounde, were evaluated for total IgG and IgA antibody levels by radial diffusion, protein composition by SDS-PAGE, anti-malarial antibodies using an isotype-specific immunofluorescence assay, and the ability to immunoprecipitate Plasmodium falciparum antigens metabolically labelled with 35S-methionine. Results showed that anti-P. falciparum antibodies were present in breast milk, and that paired milk and serum samples from individual women contained Abs that recognized similar malarial antigens.

Development of a malaria T-cell vaccine for blood stage immunity.

We have defined a strategy for the development of a T-cell vaccine for blood stage immunity, taking into consideration the central role of T cells and MHC restriction in malaria immune responses. We have used the AMPHI computer algorithm to identify putative T-cell epitopes from conserved regions of 11 Plasmodium falciparum asexual stage proteins. Ten of the eleven proteins are currently candidates for vaccine development. Using this algorithm we selected 22 putative T-cell epitope peptides and 8 control peptides. These peptides were used to test the T-cell responses of three defined populations of Caucasians who have (1) recovered from P. falciparum malaria, (2) been exposed, but never clinically infected, (3) never been exposed or infected. Preliminary analysis of our data shows population differences in T-cell responses to putative T-cell epitope peptides. Ultimately, these studies will help to identify those T epitopes that can be incorporated into a T-cell vaccine for protective immunity.

The development and validation of an enzyme linked immunosorbent assay for malaria.

ELISA has been evaluated and validated for malaria. It is sensitive, specific and reproducible. A comparison of ELISA and IFAT has been made, and there is a good correlation between the two tests. ELISA results can be assessed objectively. A small study on Caucasians with clinical malaria shows the value of ELISA as an immunodiagnostic tool. Its seroepidemiological value is also demonstrated, since it quickly reflects the changes in malaria transmission after a WHO malaria control programme in a malaria endemic area.

Antibodies to Plasmodium falciparum gamete surface antigens in Papua New Guinea sera.

Sera from individuals living in malaria endemic areas of Papua New Guinea were tested for their effect on infectivity of Plasmodium falciparum gametocytes grown in culture to Anopheles freeborni mosquitoes. Consistent reduction of infectivity to less than 5% of control was observed with nine out of the 41 sera from the endemic area tested and also with three out of seven sera tested from individuals rarely exposed to malaria infection. Gamete surface antigens recognized by the sera were investigated by immunoprecipitation from 125I surface-labelled gametes extracted in SDS and Triton X-100. The main antigens recognized were of the same mol. wt (230, 48 and 45 kD) as those known to be targets of transmission-blocking monoclonal antibodies. A significant negative correlation was observed between the total ct/min immunoprecipitated from surface-labelled gametes by the sera and the average number of oocysts per gut observed in membrane feeding experiments with these sera. Spearmann's rank correlation coefficient indicated that suppression of infectivity correlated strongly with the presence of antibodies against the 230 kD protein; there was no significant correlation between suppression and antibodies to the 48/45 kD proteins. The antibody response to the different gamete surface antigens varied greatly in sera from the endemic areas suggesting that individuals respond differently to each gamete antigen.