Search

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1–3, but also revealed how quickly viral escape can curtail effective options4, 5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including EvusheldTMand its constituent, cilgavimab4, 6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protectionin vivoagainst the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention, post-exposure prophylaxis, or therapy. However, the titer of neutralizing antibodies required for protection against SARS-CoV-2 infection remains poorly characterized. We previously described two potently neutralizing mAbs COV2-2130 and COV2-2381 targeting non-overlapping epitopes on the receptor-binding domain of SARS-CoV-2 spike protein. Here, we engineered the Fc-region of these mAbs with mutations to extend their persistence in humans and reduce interactions with Fc gamma receptors. Passive transfer of individual or combinations of the two antibodies (designated ADM03820) given prophylactically by intravenous or intramuscular route conferred virological protection in a non-human primate (NHP) model of SARS-CoV-2 infection, and ADM03820 potently neutralized SARS-CoV-2 variants of concernin vitro. We defined 6,000 as a protective serum neutralizing antibody titer in NHPs against infection for passively transferred human mAbs that acted by direct viral neutralization, which corresponded to a concentration of 20 μg/mL of circulating mAb.

The genome of Francisella tularensis live vaccine strain NR-28537 was sequenced by a hybrid approach utilizing an Oxford Nanopore Technologies R9 flow cell and an Illumina MiSeq platform. De novo assembly of the resulting long and short reads produced a single-contig whole-genome sequence.

Background Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention or therapy. The pre-exposure prophylactic efficacy of neutralizing antibodies that are engineered with mutations to extend their persistence in human serum and the neutralizing antibody titer in serum required for protection against SARS-CoV-2 infection remain poorly characterized. Methods The Fc-region of two neutralizing mAbs (COV2-2130 and COV2-2381) targeting non-overlapping epitopes on the receptor-binding domain of SARS-CoV-2 spike protein was engineered to extend their persistence in humans and reduce interactions with Fc gamma receptors. We assessed protection by individual antibodies or a combination of the two antibodies (designated ADM03820) given prophylactically by intravenous or intramuscular route in a non-human primate (NHP) model of SARS-CoV-2 infection. Findings Passive transfer of individual mAbs or ADM03820 conferred virological protection in the NHP respiratory tract in a dose-dependent manner, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined a protective serum neutralizing antibody titer and concentration in NHPs for passively transferred human antibodies that acted by direct viral neutralization. Conclusions In summary, we demonstrate that neutralizing antibodies with extended half-life and lacking Fc-mediated effector functions are efficient for pre-exposure prophylaxis of SARS-CoV-2 infection in NHPs. These results support clinical development of ADM03820 for COVID-19 prevention. Funding This research was supported by a contract from the JPEO-CBRND (W911QY-20-9-003), 20-05, the Joint Sciences and Technology Office and the Joint Program Executive Office (MCDC-16-01-002 JSTO, JPEO), DARPA grant (HR0011-18-2-0001), NIH grant (R01 AI157155), and the 2019 Future Insight Prize from Merck KGaA., Graphical Abstract, In this work, Cobb et al. described a combination of two human neutralizing antibodies, engineered with extended half-life and lacking Fc effector functions, which protected non-human primates against SARS-CoV-2 infection when given prophylactically by intravenous or intramuscular route. The serum antibody titer required for virological protection was defined.

The lipoprotein OspA is produced by the Lyme disease spirochetes primarily in unfed ticks. OspA production is down-regulated by the blood meal and it is not produced in mammals except for possible transient production during late stage infection in patients with Lyme arthritis. Vaccination with OspA elicits antibody (Ab) that can target spirochetes in the tick midgut during feeding and inhibit transmission to mammals. OspA was the primary component of the human LYMErix™ vaccine. LYMErix™ was available from 1998 to 2002 but then pulled from the market due to declining sales as a result of unsubstantiated concerns about vaccination induced adverse events and poor efficacy. It was postulated that a segment of OspA that shares sequence similarity with a region in human LFA-1 and may trigger putative autoimmune events. While evidence supporting such a link has not been demonstrated, most efforts to move forward with OspA as a vaccine component have sought to eliminate this region of concern. Here we identify an OspA linear epitope localized within OspA amino acid residues 221–240 (OspA221–240) that lacks the OspA region suggested to elicit autoimmunity. A peptide consisting of residues 221–240 was immunogenic in mice. Ab raised against OspA221–240 peptide surface labeled B. burgdorferi in IFAs and displayed potent Ab mediated-complement dependent bactericidal activity. BLAST analyses identified several variants of OspA221–240 and a closely related sequence in OspB. It is our hypothesis that integration of the OspA221–240 epitope into a multivalent-OspC based chimeric epitope based vaccine antigen (chimeritope) could result in a subunit vaccine that protects against Lyme disease through synergistic mechanisms.

Experimental Outer surface protein (Osp) C based subunit chimeritope vaccinogens for Lyme disease (LD) were assessed for immunogenicity, structure, ability to elicit antibody (Ab) responses to divergent OspC proteins, and bactericidal activity. Chimeritopes are chimeric epitope based proteins that consist of linear epitopes derived from multiple proteins or multiple variants of a protein. An inherent advantage to chimeritope vaccinogens is that they can be constructed to trigger broadly protective Ab responses. Three OspC chimeritope proteins were comparatively assessed: Chv1, Chv2 and Chv3. The Chv proteins possess the same set of 18 linear epitopes derived from 9 OspC type proteins but differ in the physical ordering of epitopes or by the presence or absence of linkers. All Chv proteins were immunogenic in mice and rats eliciting high titer Ab. Immunoblot and enzyme linked immunosorbent assays demonstrated that the Chv proteins elicit IgG that recognizes a diverse array of OspC type proteins. The panel included OspC proteins produced by N. American and European strains of the LD spirochetes. Rat anti-Chv antisera uniformly labeled intact, non-permeabilized Borreliella burgdorferi demonstrating that vaccinal Ab can bind to targets that are naturally presented on the spirochete cell surface. Vaccinal Ab also displayed potent complement dependent-Ab mediated killing activity. This study highlights the ability of OspC chimeritopes to serve as vaccinogens that trigger potentially broadly protective Ab responses. In addition to the current use of an OspC chimeritope in a canine LD vaccine, chimeritopes can serve as key components of human LD subunit vaccines.

As Ixodes ticks spread to new regions, the incidence of Lyme disease (LD) in companion animals and humans will increase. Preventive strategies for LD in canines center on vaccination and tick control (acaricides). Both subunit and bacterin based LD veterinary vaccines are available. Outer surface protein C (OspC), a potent immunogen and dominant early antigen, has been demonstrated to elicit protective antibody (Ab) responses. However, a single OspC protein elicits a relatively narrow range of protection. There are conflicting reports as to whether the immunodominant epitopes of OspC reside within variable or conserved domains. A detailed understanding of the antigenic determinants of OspC is essential for understanding immune responses to this essential virulence factor and vaccinogen. Here, we investigate the contribution of the conserved C-terminal C10 motif in OspC triggered Ab responses. Using a panel of diverse recombinant full length OspC proteins and their corresponding C10 deletion variants (OspCΔC10), we demonstrate that the C10 motif does not significantly contribute to immunization or infection induced Ab responses in rabbits, rats, canines, horses and non-human primates. Furthermore, the C10 motif is not required to trigger potent bactericidal Ab responses. This study provides insight into the antigenic structure of OspC. The results enhance our understanding of immune responses that develop during infection or upon vaccination and have implications for interpretation of LD diagnostic assays that employ OspC.

The primary causative agent of tick-borne relapsing fever in North America is Borrelia hermsii . It has been hypothesized that B. hermsii evades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement. In vitro , B. hermsii produces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen for B. hermsii infection in humans. The ability to test the hypothesis that FhbA is a critical virulence factor in vivo has been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated a B. hermsii fhbA deletion mutant (the B. hermsii YORΔ fhbA strain) through allelic exchange mutagenesis. Deletion of fhbA abolished FH binding by the YORΔ fhbA strain and eliminated cleavage of C3b on the cell surface. However, the YORΔ fhbA strain remained infectious in mice and retained resistance to killing in vitro by human complement. Collectively, these results indicate that B. hermsii employs an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n = 2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.

OspC performs essential functions during the enzootic cycle of the Lyme disease (LD) spirochetes. In this study, the specificity of antibody (Ab) responses to OspC was profiled to define the antigenic determinants during infection and after vaccination. Several OspC variants or ‘types’ were screened with serum from SNAP4Dx C6 positive dogs and with serum from rabbits hyperimmunized with OspC proteins. The OspC type-specific nature of the Ab response revealed that variable domains of OspC are immunodominant during infection and upon vaccination. To assess the potential of OspC to elicit Ab in the context of a bacterin vaccine, OspC production in strains cultivated in vitro was assessed. Immunoblot and indirect immunofluorescent antibody analyses demonstrated that production is low and that only a subset of cells actively produces OspC in vitro, raising questions about the potential of bacterin vaccines to stimulate significant anti-OspC Ab responses. The specificity of the OspC Ab response in experimentally infected mice over time was assessed to determine if domains shielded in the OspC homodimer become accessible and stimulate Ab production as infection progresses. The results demonstrate that the OspC Ab response remains focused on surface exposed variable regions of the protein throughout infection. In contrast to some earlier studies, it is concluded that conserved domains of OspC, including the C7 or C10 domain, do not elicit significant Ab responses during infection or upon vaccination. Collectively, the results indicate that OspC diversity must be considered in vaccine design and in the interpretation of diagnostic assays that employ OspC as a diagnostic antigen.

Borrelia burgdorferiOspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution amongB. burgdorferisensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization.B. burgdorferiB31ospCwas replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required forin vivofunction, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.

Lyme disease is the most common vector-borne disease in North America and Europe and, if untreated, has significant arthritic, cardiac, dermatological and neurological sequelae. There is no currently available human Lyme disease vaccine. Outer surface protein C, because of its antigenicity, protective ability, and expression characteristics has emerged as a promising second generation vaccine candidate; however, significant sequence heterogeneity has impeded its development. Analyses of OspC sequences have revealed the existence of stable phylogenetic clusters or types, and that the type-defining sequence variation occurs within defined domains of the protein. Recent data indicating that immunodominant, and potentially protective OspC epitopes are located in these hypervariable regions has allowed development of a tetravalent, epitope-based, chimeric vaccine. In this report, we have extended that previously described tetravalent construct to include four additional OspC types. We demonstrate that the construct is highly immunogenic, and elicits type-specific antibodies that recognize each of the eight incorporated OspC type-specific epitopes. Antibody raised to the octavalent construct readily binds to the surface of strains expressing each component OspC type, indicating that the incorporated epitopes are presented on the surface of intact cells. In addition, the construct elicits antibody isotypes associated with complement-dependent bactericidal activity. These results represent an important step forward in the design of a broadly protective polyvalent OspC-based Lyme disease vaccine.

There is currently no Lyme disease vaccine commercially available for use in humans. Outer surface protein C (OspC) of the Borrelia has been widely investigated as a potential vaccinogen. At least 38 OspC types have been defined. While the antibody response to OspC is protective, the range of protection is narrow due to the localization of protective epitopes within OspC type-specific domains. To develop a broadly protective vaccine, we previously constructed a tetravalent chimeric vaccinogen containing epitopes from OspC types A, B, K, and D. While this construct elicited bactericidal antibody against strains bearing each of the four OspC types, its solubility was low, and decreasing IgG titer to epitopes near the C-terminus of the construct was observed. In this report, construct solubility and immunogenicity were increased by dialysis against an Arg/Glu buffer. We also demonstrate the immunogenicity of the construct in alum. To further optimize epitope-specific immune responses, several constructs were generated with differing epitope organization or with putative C-terminal protective motifs. Analyses of murine antibody titers and isotype profiles induced by these constructs revealed that while the C-terminal tags did not enhance antibody titer, specific epitope reorganization and reiteration did. These analyses provide important information that can be exploited in the development of chimeric vaccinogens in general.

The OspC protein of Borrelia burgdorferi is an immunodominant antigen. Here we demonstrate that the loop 5 domain of type A OspC is surface exposed, elicits bactericidal antibody in mice, and is antigenic in humans. The data suggest that loop 5 may be suitable for inclusion in a polyvalent, chimeric OspC vaccinogen.

Concentrated culture supernatants containing the extracellular products (ECP) of the protozoan oyster parasite Perkinsus marinus were used to immunize mice. This preparation, produced by ultrafiltration, was found to be both poorly immunogenic and toxic to experimental animals. The possibility that these effects were due to toxic parasite products and/or medium constituents was examined. Co-administration of this material with highly immunogenic oyster hemolymph caused a substantive suppression of the specific antibody response to hemolymph, as well as a decrease in the number of epitopes recognized. Potential protein/protease toxin-mediated causes of the immunosuppression were addressed by heat denaturation and proteolytic inhibition of the concentrate; neither substantially enhanced immunogenicity. Analysis of media constituents revealed that the known immunomodulatory surfactant, Pluronic F-68 (PF68), used in the defined lipid concentrate supplement, was capable of eliciting significant immunosuppression. Although isolated protein antigens from P. marinus ECP remain highly immunosuppressive, separation of the protein antigens from the PF68 has enabled production of polyclonal antisera with a broader recognition of antigens.

Lyme disease in the United States is caused byBorrelia burgdorferisensu stricto, which is transmitted to mammals by infected ticks.Borreliaspirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses toB. burgdorferiOsp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

Tick-borne relapsing fever (TBRF) is a spirochetal disease caused by at least 15 different Borrelia species. It is a serious human health concern in regions of endemicity throughout the world. Transmission to humans occurs through the bites of infected Ornithodoros ticks. In North America, the primary Borrelia species associated with human disease are B. hermsii and B. turicatae . Direct demonstration of the role of putative TBRF spirochete virulence factors in the disease process has been hindered by the lack of a genetic manipulation system and complete genome sequences. Expanding on recent developments in these areas, here we demonstrate the successful generation of a clone of B. hermsii YOR that constitutively produces green fluorescent protein (GFP) ( B. hermsii YOR:: kan gfp ). This strain was generated through introduction of a kan-gfp cassette into a noncoding region of the 200-kb B. hermsii linear plasmid lp200. Genetic manipulation did not affect the growth rate or trigger the loss of native plasmids. B. hermsii YOR:: kan gfp retained infectivity and elicited host seroconversion. Stable production of GFP was demonstrated both in vitro and in vivo . This study represents a significant step forward in the development of tools that can be employed to study the virulence mechanisms of TBRF spirochetes.

Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1' of the apposing monomers form a solvent-accessible pocket at the dimeric interface that presents a putative ligand-binding domain (LBD1). Here we employ site-directed and allelic-exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure-functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.

Using available Borrelia outer surface protein C (OspC) sequences, a phylogenetic analysis was undertaken to delineate the number of antigenic domains required for inclusion in a broadly protective, chimeric, OspC-based Lyme disease vaccine. The data indicate that approximately 34 would be required and that an OspC-based vaccinogen is feasible.

Lyme disease is the most common arthropod-borne disease in North America and Europe. At present, there is no commercially available vaccine for use in humans. Outer surface protein C (OspC) has antigenic and expression characteristics that make it an attractive vaccine candidate; however, sequence heterogeneity has impeded its use as a vaccinogen. Sequence analyses have identified 21 well defined OspC phyletic groups or "types" (designated A-U). In this report we have mapped the linear epitopes presented by OspC types B, K, and D during human and murine infection and exploited these epitopes (along with the previously identified type A OspC linear epitopes) in the development of a recombinant, tetravalent, chimeric vaccinogen. The construct was found to be highly immunogenic in mice and the induced antibodies surface labeled in vitro cultivated spirochetes. Importantly, vaccination induced complement-dependent bactericidal antibodies against strains expressing each of the OspC types that were incorporated into the construct. These results suggest that an effective and broadly protective polyvalent OspC-based Lyme disease vaccine can be produced as a recombinant, chimeric protein.

Outer surface protein C (OspC) of the Lyme disease spirochetes is an important virulence factor that has potential utility for vaccine development. Of the 21 OspC types that have been identified, it has been postulated that types A, B, I, and K are specifically associated with invasive infections. Through an analysis of isolates collected from patients in Maryland we found that OspC types C, D, and N are also associated with invasive infections. This observation suggests that there is greater diversity in the group of OspC types associated with invasive infection than has been previously suggested. Detailed knowledge of the antigenic structure of OspC is essential for vaccine development. To determine if the antibody response to OspC is type specific, recombinant proteins of several different OspC types were immunoblotted and screened with sera from mice infected with isolates having known OspC types. These analyses revealed a high degree of specificity in the antibody response and suggested that the immunodominant epitopes of OspC reside in the variable domains of the protein. To localize these epitopes, OspC fragments were generated and screened with serum collected from infected mice. These analyses led to identification of previously uncharacterized epitopes that define the type specificity of the OspC antibody response. These analyses provide important insight into the antigenic structure of OspC and also provide a basis for understanding the variable nature of the antibody response to this important virulence factor of the Lyme disease spirochetes.

The extracellular products (ECPs) of the oyster parasite Perkinsus marinus have been posed to contain virulence factors, including serine proteases. Supplementation of P. marinus cultures with oyster homogenates enhances infectivity and changes the ECP composition. Therefore, subtractive immunization was used to attempt creation of antibodies to proteins unique to ECPs produced following parasite exposure to oyster homogenates. While control mice remained competent to respond to an unrelated antigen, no serum titers against novel ECP epitopes were detected in experimental mice. Attempts to create discriminatory hybridomas resulted in no clones with anti-ECP specificity. These findings suggest that, because no unique epitopes can be found within ECPs generated following exposure of P. marinus to host homogenates, the changes to ECPs are greatly constrained.

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus -susceptible ( Crassostrea virginica ) and -tolerant ( Crassostrea gigas , Crassostrea ariakensis ) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.