Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine.

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.

Antibodies to a single, conserved epitope in Anopheles APN1 inhibit universal transmission of Plasmodium falciparum and Plasmodium vivax malaria.

Malaria transmission-blocking vaccines (TBVs) represent a promising approach for the elimination and eradication of this disease. AnAPN1 is a lead TBV candidate that targets a surface antigen on the midgut of the obligate vector of the Plasmodium parasite, the Anopheles mosquito. In this study, we demonstrated that antibodies targeting AnAPN1 block transmission of Plasmodium falciparum and Plasmodium vivax across distantly related anopheline species in countries to which malaria is endemic. Using a biochemical and immunological approach, we determined that the mechanism of action for this phenomenon stems from antibody recognition of a single protective epitope on AnAPN1, which we found to be immunogenic in murine and nonhuman primate models and highly conserved among anophelines. These data indicate that AnAPN1 meets the established target product profile for TBVs and suggest a potential key role for an AnAPN1-based panmalaria TBV in the effort to eradicate malaria.

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

Efficacy of multivalent adenovirus-based vaccine against simian immunodeficiency virus challenge.

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

Comparative analysis of immune responses induced by vaccination with SIV antigens by recombinant Ad5 vector or plasmid DNA in rhesus macaques.

DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.

Tolerability of two sequential electroporation treatments using MedPulser DNA delivery system (DDS) in healthy adults.

Immunotherapy against infectious agents and malignant tumors requires efficient priming of effector cells through direct expression and/or efficient cross-presentation of antigens by antigen-presenting cells. Electroporation is a new procedure aimed at transiently increasing cell membrane permeability and direct delivery of antigen or antigen-encoding nucleic acids inside targeted cells. We evaluated the tolerability including compliance with repeated electroporation treatments using MedPulser DDS in 24 healthy adults. Pain severity was evaluated at time of electroporation treatment, and at 1, 5, 10, and 20 minutes, and 24 hours thereafter, using two clinically validated questionnaires: McGill Pain Questionnaire (MPQ) (Present Pain Intensity) and Brief Pain Inventory (BPI). Electroporation treatments were generally well tolerated. Twenty-two out of 24 subjects returned for the second electroporation treatment 14 days after first treatment. Only two subjects reported a treatment-related systemic adverse experience following either electroporation application. For both pain assessment tools, maximum pain and/or discomfort were mostly reported immediately (within 5 minutes) after electroporation; Furthermore, no difference was observed when comparing peak-pain scores after first and second electroporation treatments. This study supports the clinical application of MedPulser DDS for the improvement of antigen-induced immune responses for prophylactic or therapeutic vaccines, especially in gene-based therapies for cancer.

Design and optimization of a multiplex anti-influenza peptide immunoassay.

Current flu vaccines are based on killed or attenuated virus vaccines that must be altered each year to include the hemagglutinin and neuraminidase genes from a strain of virus predicted to predominate in the coming year. A vaccine that could protect against multiple strains of influenza A and B would be a major asset in the fight against flu-related mortality and morbidity. To support development of such a vaccine, we have developed a Flu Multiplex Assay based on a Luminex platform to assess serum antibody levels to two conserved peptides derived from influenza A (M2 protein) and influenza B (hemagglutinin protein). The peptides were synthesized with a biotin label and subsequently coupled to two different LumAvidin microspheres. We then tested various sera against both types of peptide in the multiplex assay format. The data show that sera from Rhesus macaques immunized with a single peptide react only with the homologous peptide while Rhesus macaques immunized with both peptides respond well to both peptides. Additionally, we were able to specifically compete reactivity to both peptides. We have tested serial bleeds from 100 pediatric patients at ages ranging from 16 to 56 weeks as well as single bleeds from over 100 healthy adults. No overall trend in titer relative to pediatric age was detected. Both demographics exhibited a minimal response to either the A/M2 or B/HA0 peptides. However, the average titer for the pediatric serum samples was significantly lower than that found in the adult population. The adult population exhibited a higher prevalence of low reactive samples. Assay reagents and parameters have been optimized and the assay is shown to be repeatable and robust. The assay will be used to support clinical vaccine trials of a bivalent peptide vaccine.

HIV type 1 vaccines for worldwide use: predicting in-clade and cross-clade breadth of immune responses.

One of the greatest challenges in HIV vaccine development is accommodating the worldwide sequence diversity of the HIV-1 virus. To understand how viral sequence diversity may affect the potential breadth of HIV-1 vaccines designed to elicit antiviral T cell immunity, we have developed novel approaches to assess sequence conservation at the amino acid level, where vaccine effects are exerted. Taking each sequence from the LANL 2004 amino acid alignments as a potential vaccine or as a challenge virus, all pairwise combinations of sequences were evaluated by two methods: first, a traditional comparison of aligned sequences, and second, by a new walking 9-mer algorithm chosen to emphasize the typical length of an MHC-I epitope. The rules for comparing mismatched 9-mer pairs between vaccine and challenge sequences were empirically deduced from an experiment on Nef-specific CD8 epitopes and the viral sequences from naturally HIV-1-infected patients. Results were weighted such that each clade contributed in proportion to its global prevalence. Cross-clade breadth of response is best maintained for vaccines encoding Pol and Gag, while commonly proposed Env- and Tat-based vaccines would be more clade sensitive. We evaluated the additional breadth that could be expected from multiclade vaccines including consensus and ancestral sequences. For more diverse proteins, adding a second strain can add a significant increase in breadth, although for three or more strains the intrinsic diversity of the protein leads to diminishing improvement.

Preclinical evaluations of peptide-conjugate vaccines targeting the antigenic domain-2 of glycoprotein B of human cytomegalovirus.

The Antigenic Domain 2 (AD-2) is a short region near the N-terminus of glycoprotein B of human cytomegalovirus (HCMV). AD-2 has been shown to contain linear epitopes that are targets for neutralizing monoclonal antibodies from human subjects with natural HCMV infection. However, AD-2 appears to be masked by the adjacent immunodominant AD-1 region. We assessed a serum panel from HCMV-seropositive individuals and found a wide range of antibody titers to AD-2; these did not correlate to serum neutralization. To expose potential epitopes in AD-2, we constructed a series of AD-2 peptide-conjugate vaccines. Mice were immunized 3 times and produced high and sustained antibody titers to AD-2 peptides, but neutralization was weak even after a single boost with whole HCMV virions. Rabbits were likewise immunized with AD-2 peptide vaccines, and produced a robust antibody response, but neutralization was inferior to a recombinant gB vaccine with an oil-in-water adjuvant. These results highlight the challenges of developing a peptide-based vaccine specific to the HCMV gB AD-2 region.

A replication-defective human cytomegalovirus vaccine for prevention of congenital infection.

Congenital human cytomegalovirus (HCMV) infection occurs in ~0.64% of infants born each year in the United States and is the leading nongenetic cause of childhood neurodevelopmental disabilities. No licensed HCMV vaccine is currently available. Natural immunity to HCMV in women before pregnancy is associated with a reduced risk of fetal infection, suggesting that a vaccine is feasible if it can reproduce immune responses elicited by natural infection. On the basis of this premise, we developed a whole-virus vaccine candidate from the live attenuated AD169 strain, with genetic modifications to improve its immunogenicity and attenuation. We first restored the expression of the pentameric gH/gL/pUL128-131 protein complex, a major target for neutralizing antibodies in natural immunity. We then incorporated a chemically controlled protein stabilization switch in the virus, enabling us to regulate viral replication with a synthetic compound named Shield-1. The virus replicated as efficiently as its parental virus in the presence of Shield-1 but failed to produce progeny upon removal of the compound. The vaccine was immunogenic in multiple animal species and induced durable neutralizing antibodies, as well as CD4+ and CD8+ T cells, to multiple viral antigens in nonhuman primates.

DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

Development of neutralizing monoclonal antibodies for oncogenic human papillomavirus types 31, 33, 45, 52, and 58.

Human papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic.

Pairwise antibody footprinting using surface plasmon resonance technology to characterize human papillomavirus type 16 virus-like particles with direct anti-HPV antibody immobilization.

This paper describes an approach to surface plasmon resonance (SPR) based epitope mapping, also referred to as pairwise antibody footprinting, involving the direct immobilization of an antigen-specific primary mAb to the surface of an SPR interface. This technique offers a more straightforward approach than indirect capture (e.g., via rabbit anti-mouse Fc) as it does not require additional steps to block the unoccupied immobilized anti-Fc to prevent non-specific antibody binding. This is also an alternative to the direct immobilization of an antigen of interest, which may cause conformational changes in the antigen or epitope degradation upon chemical immobilization, particularly in successive regeneration cycles. It is particularly suitable for highly multivalent targets such as virus-like particles (VLPs). Using this technique, we assessed a panel of eight monoclonal antibodies against HPV (human papilloma virus) L1 protein VLPs expressed by Saccharomyces cerevisiae. In the antibody epitope screening studies, HPV16 L1-directed conformational mAbs were clearly distinguished from the linear mAbs and consistent with known epitope information. Additional studies using a linear mAb and a conformational mAb demonstrate the practical application of this technique for characterizing the result of process changes and the consistency of recombinant HPV16 VLPs. The method is readily extensible to other VLPs and VLP-based vaccines.

An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform.

Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopia(TM) Epitope Discovery System(TM), which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens.

Restoration of viral epithelial tropism improves immunogenicity in rabbits and rhesus macaques for a whole virion vaccine of human cytomegalovirus.

Maternal immunity to human cytomegalovirus (HCMV) prior to conception is ~70% protective against congenital transmission and in utero infection of HCMV. Both functional antibodies capable of neutralizing virus and effective T-cells are believed to be important for the protection. Previous HCMV vaccines have rarely been shown able to induce neutralizing antibody titers comparable to those seen in naturally infected HCMV seropositive subjects. Recent studies link a glycoprotein H (gH) complex to receptor-mediated viral entry of endothelial/epithelial cells and leukocytes. This pentameric gH complex, composed of five proteins (gH, gL, UL128, UL130 and UL131 proteins), is notably missing in all HCMV vaccine previously evaluated in clinic. Here we showed that a HCMV virus, with restored expression of the pentameric gH complex, can induce 10-fold higher neutralizing antibody titers than an attenuated AD169 virus or a recombinant glycoprotein B vaccine in multiple animal species in which viral replication is not expected. Encouragingly, the peak neutralizing titers post vaccination in rabbits and monkeys were within 2-4-fold of the levels determined in HCMV seropositive subjects. Functional antibodies by vaccination could further be improved when formulated with a novel adjuvant, and the titers of the antiviral antibodies were sustained in rabbits for over a year after vaccination. These results indicate that the pentameric gH complex is associated with greatly improved functional antibodies following vaccination, and support a vaccine concept based on a nonreplicating whole HCMV with the pentameric gH-associated epithelial tropism restored.

A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines.

Neutralizing antibodies are considered an important component of protective immunity against congenital infection of human cytomegalovirus (HCMV), a frequently cited cause of birth defects. An effective HCMV vaccine is desired to induce potent neutralizing antibodies in seronegative females, so that the viral transmission to fetus would be blocked if the women contracted HCMV infections during their pregnancies. We describe a novel microneutralization assay to measure antiviral activities against HCMV in serum samples. The assay is based on detection of a dominant HCMV antigen expressed in cells, using near infrared dye-labeled immune reagents. Since the detection is independent of viral cytopathic effects, this assay format has the appeal of a short turn-around time and a read-out that is not subject to operator experience and judgment, making it a promising platform to support large scale clinical studies. In a serological survey of a cohort of 200 healthy females, we showed that the neutralizing titers measured in this assay are highly comparable to those from a neutralization assay based on an enzyme-linked immunostaining method. Lastly, to demonstrate the utility of this assay to support HCMV vaccine study, we presented the results of neutralizing titers from a rhesus macaque vaccination study.

Quantitative analysis of neutralizing antibody response to human cytomegalovirus in natural infection.

Naturally acquired immunity significantly reduces the risk of congenital cytomegalovirus (CMV) infection in future pregnancies. An immune response comparable to that of natural infection has been used as a benchmark for CMV vaccine efficacy; however, the magnitude and persistence of the neutralizing antibody responses in naturally infected women are not completely understood. In this study, we quantitatively analyzed a panel of 375 female CMV convalescent sera ranging in age from 18 to 84 years, for its ability to block virus entry into epithelial cells and fibroblasts, as well as its binding potential to CMV particles. The geometric mean titer of the sera in this panel to neutralize 50% of the virus entry into epithelial cells was 7491, compared to 802 for entry into fibroblasts. The epithelial neutralizing titers were statistically indistinguishable among different age groups, and conformed to a normal distribution. There was a weak correlation between the levels of neutralization and the binding activities to viral particles. Our data confirmed that natural CMV infection in healthy women induces potent neutralizing antibodies against infection of both fibroblasts and epithelial cells. The serum neutralizing activities were maintained at high levels throughout the child bearing age. The corresponding titers may serve as a biomarker for CMV vaccine efficacy.

Mapping HIV-1 vaccine induced T-cell responses: bias towards less-conserved regions and potential impact on vaccine efficacy in the Step study.

UNLABELLED: T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. TRIAL REGISTRATION: ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.