Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ.

The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1-response-inducing adjuvant when formulated in a squalene oil-in-water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA-SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA-SE, in mice. Within the innate response to adjuvants, only GLA-SE displayed features of inflammasome activation, evidenced by early IL-18 secretion and IFN-γ production in memory CD8(+) T cells and neutrophils. Such early IFN-γ production was ablated in caspase-1/11(-/-) mice and in IL-18R1(-/-) mice. Furthermore, caspase-1/11 and IL-18 were also required for full Th1 CD4(+) T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-in-water based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists.

Protection against Mycobacterium leprae infection by the ID83/GLA-SE and ID93/GLA-SE vaccines developed for tuberculosis.

Despite the dramatic reduction in the number of leprosy cases worldwide in the 1990s, transmission of the causative agent, Mycobacterium leprae, is still occurring, and new cases continue to appear. New strategies are required in the pursuit of leprosy elimination. The cross-application of vaccines in development for tuberculosis may lead to tools applicable to elimination of leprosy. In this report, we demonstrate that the chimeric fusion proteins ID83 and ID93, developed as antigens for tuberculosis (TB) vaccine candidates, elicited gamma interferon (IFN-γ) responses from both TB and paucibacillary (PB) leprosy patients and from healthy household contacts of multibacillary (MB) patients (HHC) but not from nonexposed healthy controls. Immunization of mice with either protein formulated with a Toll-like receptor 4 ligand (TLR4L)-containing adjuvant (glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) stimulated antigen-specific IFN-γ secretion from pluripotent Th1 cells. When immunized mice were experimentally infected with M. leprae, both cellular infiltration into the local lymph node and bacterial growth at the site were reduced relative to those of unimmunized mice. Thus, the use of the Mycobacterium tuberculosis candidate vaccines ID83/GLA-SE and ID93/GLA-SE may confer cross-protection against M. leprae infection. Our data suggest these vaccines could potentially be used as an additional control measure for leprosy.

A defined subunit vaccine that protects against vector-borne visceral leishmaniasis.

Vaccine development for vector-borne pathogens may be accelerated through the use of relevant challenge models, as has been the case for malaria. Because of the demonstrated biological importance of vector-derived molecules in establishing natural infections, incorporating natural challenge models into vaccine development strategies may increase the accuracy of predicting efficacy under field conditions. Until recently, however, there was no natural challenge model available for the evaluation of vaccine candidates against visceral leishmaniasis. We previously demonstrated that a candidate vaccine against visceral leishmaniasis containing the antigen LEISH-F3 could provide protection in preclinical models and induce potent T-cell responses in human volunteers. In the present study, we describe a next generation candidate, LEISH-F3+, generated by adding a third antigen to the LEISH-F3 di-fusion protein. The rationale for adding a third component, derived from cysteine protease (CPB), was based on previously demonstrated protection achieved with this antigen, as well as on recognition by human T cells from individuals with latent infection. Prophylactic immunization with LEISH-F3+formulated with glucopyranosyl lipid A adjuvant in stable emulsion significantly reduced both Leishmania infantum and L. donovani burdens in needle challenge mouse models of infection. Importantly, the data obtained in these infection models were validated by the ability of LEISH-F3+/glucopyranosyl lipid A adjuvant in stable emulsion to induce significant protection in hamsters, a model of both infection and disease, following challenge by L. donovani-infected Lutzomyia longipalpis sand flies, a natural vector. This is an important demonstration of vaccine protection against visceral leishmaniasis using a natural challenge model.

Optimizing Immunization Strategies for the Induction of Antigen-Specific CD4 and CD8 T Cell Responses for Protection against Intracellular Parasites.

Immunization strategies that generate either CD4 or CD8 T cell responses are relatively well described, but less is known with regard to optimizing regimens to induce both CD4 and CD8 memory T cells. Considering the importance of both CD4 and CD8 T cells in the control of intracellular pathogens such as Leishmania donovani, we wanted to identify vaccines that could raise both CD4 and CD8 T cell responses and determine how to configure immunization strategies to generate the best combined protective T cell response. We examined responses generated against the Leishmania vaccine antigen F3 following its administration in either recombinant form with the Toll-like receptor 4 (TLR4) agonist-containing adjuvant formulation GLA-SE (F3+GLA-SE) or as a gene product delivered in an adenoviral vector (Ad5-F3). Homologous immunization strategies using only F3+GLA-SE or Ad5-F3 preferentially generated either CD4 or CD8 T cells, respectively. In contrast, heterologous strategies generated both antigen-specific CD4 and CD8 T cells. Administration of F3+GLA-SE before Ad5-F3 generated the greatest combined CD4 and CD8 responses. Cytotoxic CD8 T cell responses were highest when Th1 cells were generated prior to their induction by Ad5-F3. Finally, a single immunization with a combination of F3+GLA-SE mixed with Ad5-F3 was found to be sufficient to provide protection against experimental L. donovani infection. Taken together, our data delineate immunization regimens that induce antigen-specific CD4 and CD8 T cell memory responses, and identify a single immunization strategy that could be used to rapidly provide protection against intracellular pathogens in regions where access to health care is limited or sporadic.

Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs.

From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE.

Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L. donovani and L. infantum in mice, measured as significant reductions in liver parasite burdens. A robust immune response to each component of the vaccine with polyfunctional CD4 TH1 cell responses characterized by production of antigen-specific interferon-γ, tumor necrosis factor and interleukin-2 (IL-2), and low levels of IL-5 and IL-10 was induced in immunized mice. We also demonstrate that CD4 T cells, but not CD8 T cells, are sufficient for protection against L. donovani infection in immunized mice. Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States. The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data. These data provide a strong rationale for additional trials in Leishmania-endemic countries in populations vulnerable to VL.

Elimination of the cold-chain dependence of a nanoemulsion adjuvanted vaccine against tuberculosis by lyophilization.

Next-generation rationally-designed vaccine adjuvants represent a significant breakthrough to enable development of vaccines against challenging diseases including tuberculosis, HIV, and malaria. New vaccine candidates often require maintenance of a cold-chain process to ensure long-term stability and separate vials to enable bedside mixing of antigen and adjuvant. This presents a significant financial and technological barrier to worldwide implementation of such vaccines. Herein we describe the development and characterization of a tuberculosis vaccine comprised of both antigen and adjuvant components that are stable in a single vial at sustained elevated temperatures. Further this vaccine retains the ability to elicit both antibody and TH1 responses against the vaccine antigen and protect against experimental challenge with Mycobacterium tuberculosis. These results represent a significant breakthrough in the development of vaccine candidates that can be implemented throughout the world without being hampered by the necessity of a continuous cold chain or separate adjuvant and antigen vials.

Development of a high density hemagglutinin protein microarray to determine the breadth of influenza antibody responses.

We have developed an influenza hemagglutinin protein microarray to assess humoral recognition of diverse influenza strains induced by vaccination and infection. Each array consists of controls and 127 hemagglutinin antigens from 60 viruses, spotted in replicates to generate a single array of 1296 spots. Six arrays are configured on a single slide, which in the following analysis was probed simultaneously with 2 isotype-specific fluorescent secondary antibodies yielding over 15,000 data points per slide. Here we report the use of this system to evaluate mouse, ferret, and human sera. The array allows simultaneous examination of the magnitude of antibody responses, the isotype of such antibodies, and the breadth of influenza strain recognition. We are advancing this technology as a platform for rapid, simple, high-throughput assessment of homologous and heterologous antibody responses to influenza disease and vaccination.

Protection of mice from Mycobacterium tuberculosis by ID87/GLA-SE, a novel tuberculosis subunit vaccine candidate.

Tuberculosis is a major health concern. Non-living tuberculosis (TB) vaccine candidates may not only be safer than the current vaccine (BCG) but could also be used to boost BCG to enhance or elongate protection. No subunit vaccines, however, are currently available for TB. To address this gap and to improve the global TB situation, we have generated a defined subunit vaccine by genetically fusing the genes of 3 potent protein Mtb antigens, Rv2875, Rv3478 and Rv1886, into a single product: ID87. When delivered with a TLR4 agonist-based adjuvant, GLA-SE, ID87 immunization reduced Mtb burden in the lungs of experimentally infected mice. The reduction in bacterial burden of ID87/GLA-SE immunized mice was accompanied by an early and significant leukocyte infiltration into the lungs during the infectious process. ID87/GLA-SE appears to be a promising new vaccine candidate that warrants further development.