Gary A. Fahle

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.

Prior Infection and Passive Transfer of Neutralizing Antibody Prevent Replication of Severe Acute Respiratory Syndrome Coronavirus in the Respiratory Tract of Mice

ABSTRACT Following intranasal administration, the severe acute respiratory syndrome (SARS) coronavirus replicated to high titers in the respiratory tracts of BALB/c mice. Peak replication was seen in the absence of disease on day 1 or 2, depending on the dose administered, and the virus was cleared within a week. Viral antigen and nucleic acid were detected in bronchiolar epithelial cells during peak viral replication. Mice developed a neutralizing antibody response and were protected from reinfection 28 days following primary infection. Passive transfer of immune serum to naïve mice prevented virus replication in the lower respiratory tract following intranasal challenge. Thus, antibodies, acting alone, can prevent replication of the SARS coronavirus in the lung, a promising observation for the development of vaccines, immunotherapy, and immunoprophylaxis regimens.

Replication of SARS coronavirus administered into the respiratory tract of African Green, rhesus and cynomolgus monkeys

Abstract SARS coronavirus (SARS-CoV) administered intranasally and intratracheally to rhesus, cynomolgus and African Green monkeys (AGM) replicated in the respiratory tract but did not induce illness. The titer of serum neutralizing antibodies correlated with the level of virus replication in the respiratory tract (AGM>cynomolgus>rhesus). Moderate to high titers of SARS-CoV with associated interstitial pneumonitis were detected in the lungs of AGMs on day 2 and were resolving by day 4 post-infection. Following challenge of AGMs 2 months later, virus replication was highly restricted and there was no evidence of enhanced disease. These species will be useful for the evaluation of the immunogenicity of candidate vaccines, but the lack of apparent clinical illness in all three species, variability from animal to animal in level of viral replication, and rapid clearance of virus and pneumonitis in AGMs must be taken into account by investigators considering the use of these species in efficacy and challenge studies.

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.

The use of oral washes to diagnose Pneumocystis carinii pneumonia: A blinded prospective study using a polymerase chain reaction-based detection system

Pneumocystis carinii pneumonia (PCP) can be diagnosed by direct microscopic examination of induced sputum or by bronchoalveolar lavage (BAL). However, many institutions have little diagnostic success with induced sputum, and BAL is invasive and expensive. This prospective, blinded study assessed oral washes as a more convenient specimen than either sputum or BAL fluid and used a dissociation-enhanced lanthanide fluoroimmunoassay time-resolved fluorescent hybridization polymerase chain reaction (PCR) detection system that is feasible for clinical laboratories. The study assessed 175 oral washes, each paired with either an induced sputum that was positive for Pneumocystis or a BAL sample. The PCR test based on the Pneumocystis major surface glycoprotein primers had a sensitivity of 91% and a specificity of 94%, compared with a test based on mitochondrial large subunit rRNA primers, which had a sensitivity of 75% and a specificity of 96%. These results suggest that oral washes can provide a useful sample for diagnosis of PCP when a sensitive PCR detection system is used.

Clinical Trial of Quantitative Real-Time Polymerase Chain Reaction for Detection of Cytomegalovirus in Peripheral Blood of Allogeneic Hematopoietic Stem-Cell Transplant Recipients

The preemptive therapy of cytomegalovirus (CMV) reactivation is useful for the prevention of CMV disease in allogeneic hematopoietic stem-cell transplant (HSCT) recipients. We compared results of the pp65 CMV antigenemia test with quantitative touch-down polymerase chain reaction (Q-PCR) on unfractionated whole blood for the detection of CMV reactivation in 51 HSCT recipients. Forty episodes of reactivation in 28 patients were detected by antigenemia and treated by antiviral drugs. Q-PCR detected CMV DNA in 39 (97.5%) of 40 reactivation episodes. False-positive results occurred in 3% of tests, of which 63% were borderline positive. Q-PCR results were positive earlier than antigenemia results in 30 (77%) of 39 episodes detected by antigenemia. Q-PCR remained positive after treatment was discontinued in 14 (36%) of 39 episodes and predicted the return of CMV reactivation in 4 (31%) of 13 episodes. Q-PCR was more sensitive than the antigenemia test and had sufficient specificity for clinical use.

Human herpes 6 virus encephalitis complicating allogeneic hematopoietic stem cell transplantation

Objective: To describe the presentation and management of encephalitis due to human herpes 6 virus (HHV-6) in patients who underwent allogeneic hematopoietic stem cell transplant (alloHSCT), via retrospective chart review. Methods: Of the 243 patients who underwent alloHSCT at the NIH Clinical Center during 2009 to 2011, we retrospectively analyzed 9 diagnosed with HHV-6 encephalitis post-alloHSCT. Results: Eight men and 1 woman (aged 19–60 years) met diagnostic criteria for study inclusion. The median time from HSCT to initial symptoms was 21 days. All patients presented with altered mental status and headaches. Seven patients had amnesia and 2 presented with fever of unknown etiology. Four patients had clinical seizures during the disease course. Brain MRI within 7 days was normal in all patients. Repeat MRI after 7 days showed hyperintensity in the limbic area in 3 patients. On initial testing, CSF analysis indicated acellularity and normal or minimally elevated protein; presence of HHV-6 was detected by PCR. After 7 days, mildly elevated protein and minimal pleocytosis were noted. Ganciclovir, foscarnet, or valganciclovir alone or in combination was initiated with subsequent improvement. Four patients remained alive at 1 year posttransplant; 2 had persistent memory deficits. Presence of encephalitis was associated with higher mortality post-alloHSCT. Conclusion: High clinical suspicion and CSF PCR testing are important for early diagnosis of HHV-6 encephalitis post-HSCT. Abnormalities on brain MRI or CSF testing may be minimal and delayed. Diagnosis and management of HHV-6 encephalitis is challenging, and a larger prospective study is needed for further research.

Toward Molecular Parasitologic Diagnosis: Enhanced Diagnostic Sensitivity for Filarial Infections in Mobile Populations

ABSTRACT The diagnosis of filarial infections among individuals residing in areas where the disease is not endemic requires both strong clinical suspicion and expert training in infrequently practiced parasitological methods. Recently developed filarial molecular diagnostic assays are highly sensitive and specific but have limited availability and have not been closely evaluated for clinical use outside populations residing in areas of endemicity. In this study, we assessed the performance of a panel of real-time PCR assays for the four most common human filarial pathogens among blood and tissue samples collected from a cohort of patients undergoing evaluation for suspected filarial infections. Compared to blood filtration, real-time PCR was equally sensitive for the detection of microfilaremia due to Wuchereria bancrofti (2 of 46 samples positive by both blood filtration and PCR with no discordant results) and L oa loa (24 of 208 samples positive by both blood filtration and PCR, 4 samples positive by PCR only, and 3 samples positive by blood filtration only). Real-time PCR of skin snip samples was significantly more sensitive than microscopic examination for the detection of Onchocerca volvulus microfiladermia (2 of 218 samples positive by both microscopy and PCR and 12 samples positive by PCR only). The molecular assays required smaller amounts of blood and tissue than conventional methods and could be performed by laboratory personnel without specialized parasitology training. Taken together, these data demonstrate the utility of the molecular diagnosis of filarial infections in mobile populations.

Lack of association between HSV-1 DNA in the brain, Alzheimer’s disease and apolipoprotein E4

An association between herpes simplex type 1 (HSV-1), ApoE4 and AD has been reported (Itzhaki et a l., 1997). HSV-1 DNA was detected by the polymerase chain reaction (PCR) in the brains of 28/44 controls and 34/46 AD patients. The odds ratio for the presence of ApoE4 allele was 16.8 in HSV-1 positive AD ve rsus 1.67 for HSV-1 negative AD. These ® ndings stimulated our own investigation of the association of HSV-1 and AD. AD patients and controls brains were provided by Dr Juan Troncoso from the Johns Hopkins University Alzheimers Disease Research Center, supported by NIH grant AG 05146. The pathological diagnosis of AD followed the recommendations of the Consortium to Establish a Registry for Alzhei-mers Disease (Mirra e t a l., 1991). The mean age (and ranges) of AD and controls were 79 (56 ± 93) and 79 (59 ± 93), respectively. All patients and controls were Caucasian. Autopsies were performed a maximum of 19 h post-mortem, with a mean post mortem delay of 9.5 h for AD and 9.2 h for controls.

Florid CD4+, CD56+ T-cell infiltrate associated with Herpes simplex infection simulating nasal NK-/T-cell lymphoma

We report a case of Herpes simplex virus (HSV) infection of the nasopharynx associated with a dense CD4+, CD56+ T-cell infiltrate that simulated lymphoma on clinical, histologic, and immunophenotypic grounds. Histologic examination showed a tumorlike lymphoid infiltrate with extensive necrosis. Multinucleated giant cells with “ground-glass” nuclei characteristic of HSV were observed in necrotic areas but were not prominent. Immunohistochemical studies of the lymphoid infiltrate revealed a predominance of T cells, positive for CD3, CD4, CD5, and CD56. Immunohistochemical staining with HSV antibody was focally positive in the multinucleated giant cells. Molecular studies using PCR and Southern blot were positive for HSV Type II. PCR studies for T-cell receptor gamma and immunoglobulin heavy chain gene rearrangements showed no evidence of a clonal population. In situ hybridization studies for Epstein–Barr virus (EBV) were negative. The clinical presentation of a large fungating mass, the extent of the lymphoid infiltrate, and the expression of CD56 all raised the possibility of a nasal NK/T cell lymphoma. However, the presence of HSV, lack of angioinvasion and angiodestruction, absence of EBV, and polyclonal T-cell nature of the infiltrate argued against this diagnosis. Although prior studies have not fully characterized the immunophenotypic features of the lymphocyte response to HSV in infected tissues, we postulate that the CD56+, CD4+ T-cell reaction represents a florid antiviral immune response.