Blockade of CCR4 breaks immune tolerance in chronic hepatitis B patients by modulating regulatory pathways.

BACKGROUND: Immunotargets including checkpoint inhibitors and toll-like receptor 8 agonists have recently gained attention for the recovery of hepatitis B virus (HBV)-specific T cell exhaustion in chronic hepatitis B(CHB). Chemokine receptors have a similar significant role during viral infections; however, their role in CHB remains poorly understood. Therefore, in this study we evaluated the role of chemokine receptor 4 (CCR4) in deriving immunosuppression during CHB. METHODS: We characterized CCR4+CD8+ T cells in CHB and identified their involvement in immunosuppression. Further, we examined if CCR4 blockade with mogamulizumab antibody can recover the functional exhaustion in HBsAg-specific T cells. RESULTS: CHB patients exhibit higher frequency of CCR4+CD8+ T cells that increase with higher HBsAg levels and fibrosis scores. In vitro, HBs antigen triggers CCR4 expression. These cells express multiple inhibitory receptors and exhibit immunosuppressive functions by producing excessive immunoregulatory cytokines IL-4, IL-5, IL-10 and TGF-ß1. CCR4 Blockade significantly boosted HBsAg-specific antiviral-cytokine production(IFN-γ, TNF-α and IL-21) in T cells through enhancing their proliferation capacity and polarizing these cells towards T helper 1(Th1) and T follicular helper cells(TFH) in case of CD4 cells, and cytotoxic T cell 1(TC1) and cytotoxic T follicular(TCF) cells in case of CD8. Cytotoxic potential was improved, while no induction of immunosuppressive-cytokines was seen after anti-CCR4 treatment thereby eliminating the risk of treatment-induced immunosuppression. CCR4 blockade inhibited the development and effector function of Tregs by controlling their expansion and TGF-ß1 production preventing Tregs-induced immunotolearance. CONCLUSIONS: CCR4 blockade reconstitutes antiviral immune response in T cells and limits the immunosuppressive functions of Tregs, representing them as a promising immunotherapeutic target for functional cure of CHB.

A Phase 2 open label study of ledipasvir/sofosbuvir for 12 weeks in subjects with hepatitis B virus infection.

Retrospective data showed that when we administered ledipasvir/sofosbuvir (LDV/SOF) to patients with hepatitis B and C coinfection, there was a modest reduction in hepatitis B surface antigen (HBsAg). Therefore, we hypothesize that similar HBsAg reduction can be seen in hepatitis B virus (HBV) monoinfected subjects. Primary and secondary efficacy endpoints are the decline in HBsAg and HBV DNA at Week 12 from baseline, respectively. We conducted an open-label Phase 2 pilot study to evaluate the safety, tolerability, and antiviral activity of LDV and/or SOF for HBV. Eligible subjects were either suppressed on antivirals (Group B) or inactive chronic HBV (Group A, C, D). Group A and B received LDV/SOF. Group C and D received SOF 400 mg and LDV 90 mg, respectively. All subjects completed the study, and all related adverse events (AEs) were mild. No discontinuations due to AEs or hepatitis flare occurred. At Week 12, HBsAg decline (log10 IU/ml) was similar between Group A (0.399) and B (0.400), less in Group C (0.207), and none in Group D, and there was HBV DNA decline in the inactive chronic HBV groups. LDV and SOF are safe and well tolerated when given to chronic hepatitis B subjects and have modest antiviral activity, particularly when given in combination.

COVID-19 Vaccines-All You Want to Know.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic has led to an unprecedented public health crisis. The collective global response has led to production of multiple safe and effective vaccines utilizing novel platforms to combat the virus that have propelled the field of vaccinology forward. Significant challenges to universal vaccine effectiveness remain, including immune evasion by SARS-CoV-2 variants, waning of immune response, inadequate knowledge of correlates of protection, and dosing in special populations. This review serves as a detailed evaluation of the development of the current SARS-CoV-2 vaccines, their effectiveness, and challenges to their deployment as a preventive tool.

Increased risk of severe COVID-19 in pregnancy in a multicenter propensity score-matched study.

OBJECTIVES: To explore the association between COVID-19 severity and pregnancy using measures such as COVID-19 ordinal scale severity score, hospitalization, intensive care unit (ICU) admission, oxygen supplementation, invasive mechanical ventilation, and death. METHODS: We conducted a retrospective, multicenter cohort study to understand the association between COVID-19 severity and pregnancy. We reviewed consecutive charts of adult females, ages 18-45, with laboratory testing for SARS-CoV-2 infection between March 1, 2020, and August 31, 2020. Cases were patients diagnosed with COVID-19 during pregnancy, whereas controls were not pregnant at the time of COVID-19 diagnosis. Primary endpoints were the COVID-19 severity score at presentation (within four hours) and the nadir of the clinical course. The secondary endpoints were the proportion of patients requiring hospitalization, ICU admission, oxygen supplementation, invasive mechanical ventilation, and death. RESULTS: A higher proportion of pregnant women had moderate to severe COVID-19 disease at the nadir of the clinical course than non-pregnant women (25 vs. 16.1 %, p=0.04, respectively). There was a higher rate of hospitalization (25.6 vs. 17.2 %), ICU admission (8.9 vs. 4.4 %), need for vasoactive substances (5.0 vs. 2.8 %), and invasive mechanical ventilation (5.6 vs. 2.8 %) in the pregnant cohort. These differences were not significant after applying propensity score matching.We found a high rate of pregnancy complications in our population (40.7 %). The most worrisome is the rate of hypertensive disorders of pregnancy (20.1 %). CONCLUSIONS: In our propensity score-matched study, COVID-19 in pregnancy is associated with an increased risk of disease severity and pregnancy complications.

Coronavirus Disease 2019-Associated Mucormycosis: Risk Factors and Mechanisms of Disease.

The severe surge of coronavirus disease 2019 (COVID-19) cases on the Indian subcontinent in early 2021 was marked by an unusually high number of COVID-19-associated mucormycosis (CAM) cases reported during this same period. This is significantly higher than predicted based on available data about prevalence and risk factors for this condition. This may be due to an unusual alignment of multiple risk factors for this condition. There is high background prevalence of mucormycosis in India likely from a high prevalence of risk factors, including undiagnosed or poorly controlled diabetes. COVID-19-induced immune dysregulation and immune suppression from steroid therapy increase the risk. The role of environmental exposure is unclear. System factors such as lack of access to healthcare during a pandemic may result in delayed diagnosis or suboptimal management with potentially poor outcomes. Here, we review currently identified risk factors and pathogenesis of CAM in a pandemic surge.

Short-duration treatment with the novel non-nucleoside inhibitor CDI-31244 plus sofosbuvir/velpatasvir for chronic hepatitis C: An open-label study.

Combination regimens of direct-acting antiviral agents (DAAs) for chronic genotype 1 hepatitis C virus (HCV) infection given for 8 or 12 weeks have high cure rates. Shortened treatment durations that maintain high cure rates may lessen treatment barriers related to affordability and drug adherence. We enrolled 12 treatment-naïve adults with chronic genotype 1 HCV infection without cirrhosis in a single-center, open-label trial to receive 2 weeks of the highly potent and selective non-nucleoside inhibitor (NNI) CDI-31244 concurrent with 6 weeks of sofosbuvir/velpatasvir. The main efficacy endpoints were sustained virologic response at 12 (SVR12) and 24 (SVR24) weeks after treatment completion. In all patients, plasma HCV RNA levels rapidly decreased during the first 2 days of treatment and were below the lower limit of quantification by the end of the 6-week treatment period. Eight of 12 (67%) patients achieved both SVR12 and SVR24. Four patients had virological relapse at Week 10, 4 weeks after end of treatment. The most common adverse event was headache, occurring in five (42%) patients. Pharmacokinetic analysis showed no relevant drug interactions between CDI-31244, sofosbuvir, and velpatasvir. In this pilot study of short-duration combination therapy involving a novel NNI with a fixed-combination DAA, 8 of 12 treatment-naïve patients with chronic genotype 1 HCV infection without cirrhosis achieved virologic cure. Future trials might evaluate whether extending the NNI duration beyond 2 weeks with combination DAAs results in higher cure rates comparable with currently approved longer duration therapy.

Immunopathology of Chronic Hepatitis B Infection: Role of Innate and Adaptive Immune Response in Disease Progression.

More than 250 million people are living with chronic hepatitis B despite the availability of highly effective vaccines and oral antivirals. Although innate and adaptive immune cells play crucial roles in controlling hepatitis B virus (HBV) infection, they are also accountable for inflammation and subsequently cause liver pathologies. During the initial phase of HBV infection, innate immunity is triggered leading to antiviral cytokines production, followed by activation and intrahepatic recruitment of the adaptive immune system resulting in successful virus elimination. In chronic HBV infection, significant alterations in both innate and adaptive immunity including expansion of regulatory cells, overexpression of co-inhibitory receptors, presence of abundant inflammatory mediators, and modifications in immune cell derived exosome release and function occurs, which overpower antiviral response leading to persistent viral infection and subsequent immune pathologies associated with disease progression towards fibrosis, cirrhosis, and hepatocellular carcinoma. In this review, we discuss the current knowledge of innate and adaptive immune cells transformations that are associated with immunopathogenesis and disease outcome in CHB patients.

RNA Interference Therapeutics for Chronic Hepatitis B: Progress, Challenges, and Future Prospects.

Chronic hepatitis B (CHB) is a global health challenge that can result in significant liver-related morbidity and mortality. Despite a prophylactic vaccine being available, patients already living with CHB often must engage in lifelong therapy with nucleoside analogues. However, the potential of RNA interference (RNAi) therapeutics as a promising avenue for CHB treatment is being explored. RNAi, particularly using small interfering RNA (siRNA), targets viral RNA that can be used to inhibit hepatitis B virus (HBV) replication. Several candidates are currently being studied and have exhibited varying success in reducing hepatitis B surface antigen (HBsAg) levels, with some showing sustained HBsAg loss after cessation of therapy. The dynamic evolution of RNAi therapy presents a promising trajectory for the development of effective and sustained treatments for CHB. This review highlights recent findings on RNAi therapeutics, including modifications for stability, various delivery vectors, and specific candidates currently in development.

Efficacy and immunogenicity following dengue virus-1 human challenge after a tetravalent prime-boost dengue vaccine regimen: an open-label, phase 1 trial.

BACKGROUND: Dengue human infection models (DHIMs) are important tools to down-select dengue vaccine candidates and establish tetravalent efficacy before advanced clinical field trials. We aimed to provide data for the safety and immunogenicity of DHIM and evaluate dengue vaccine efficacy. METHODS: We performed an open-label, phase 1 trial at the University of Maryland (Baltimore, MD, USA). Eligible participants were healthy individuals aged 18-50 years who either previously received a tetravalent dengue purified inactivated vaccine prime followed by a live-attenuated vaccine boost (ie, the vaccinee group), or were unvaccinated flavivirus-naive participants (ie, the control group). Participants in the vaccinee group with detectable pre-challenge dengue virus-1 neutralising antibody titres and flavivirus-naive participants in the control group were inoculated with dengue virus-1 strain 45AZ5 in the deltoid region, 27-65 months following booster dosing. These participants were followed-up from days 4-16 following dengue virus-1 live virus human challenge, with daily real-time quantitative PCR specific to dengue virus-1 RNA detection, and dengue virus-1 solicited local and systemic adverse events were recorded. The primary outcomes were safety (ie, solicited local and systemic adverse events) and vaccine efficacy (ie, dengue virus-1 RNAaemia) following dengue challenge. This study is registered with ClinicalTrials.gov, number NCT04786457. FINDINGS: In January 2021, ten eligible participants were enrolled; of whom, six (60%) were in the vaccinee group and four (40%) were in the control group. Daily quantitative PCR detected dengue virus-1 RNA in nine (90%) of ten participants (five [83%] of six in the vaccinee group and all four [100%] in the control group). The mean onset of RNAaemia occurred on day 5 (SD 1·0) in the vaccinee group versus day 8 (1·5) in the control group (95% CI 1·1-4·9; p=0·007), with a trend towards reduced RNAaemia duration in the vaccinee group compared with the control group (8·2 days vs 10·5 days; 95% CI -0·08 to 4·68; p=0·056). Mild-to-moderate symptoms (nine [90%] of ten), leukopenia (eight [89%] of nine), and elevated aminotransferases (seven [78%] of nine) were commonly observed. Severe adverse events were detected only in the vaccinee group (fever ≥38·9°C in three [50%] of six, headache in one [17%], and transient grade 4 aspartate aminotransferase elevation in one [17%]). No deaths were reported. INTERPRETATION: Participants who had tetravalent dengue purified inactivated vaccine prime and live-attenuated vaccine boost were unprotected against dengue virus-1 infection and further showed increased clinical, immunological, and transcriptomic evidence for inflammation potentially mediated by pre-existing infection-enhancing antibodies. This study highlights the impact of small cohort, human challenge models studying dengue pathogenesis and downstream vaccine development. FUNDING: Military Infectious Disease Research Program and Medical Technology Enterprise Consortium and Advanced Technology International.

A rare case of Sphingomonas paucimobilis ventriculitis.

Introduction: Nosocomial ventriculitis is a severe infection that habitually plagues neurological intensive care units. It is usually associated with external ventricular drains. Unfortunately, classic cerebral spinal fluid parameters are less specific and sensitive compared to community acquired meningitis. This is in part secondary to indolent bacteria commonly infecting external ventricular drains leading to ventriculitis. Case report: Herein, a rare case of Sphingomonas paucimobilis ventriculitis in an immunocompetent host is reported. The patient had classic symptoms of ventriculitis, but her cerebral spinal fluid parameters were benign and initial cultures were negative. Consequently, treatment was tailored to an assumed respiratory infection only to have recurrence of her symptoms. Repeat analysis of her cerebral spinal fluid was again benign, but her cerebral spinal fluid culture grew S. paucimobilis. Subsequently, the patient was treated with cefepime, which resolved her symptoms. She completed a two-week course and has had no recurrence of her infection. Conclusions: This case reinforces the need for clinicians to have heightened awareness of this emerging pathogen, its antibiotic resistance patterns, and the unique composition of this bacterium's cell wall which has ramifications on disease presentation.

A Comparison of Lymphoid and Myeloid Cells Derived from Human Hematopoietic Stem Cells Xenografted into NOD-Derived Mouse Strains.

Humanized mice are an invaluable tool for investigating human diseases such as cancer, infectious diseases, and graft-versus-host disease (GvHD). However, it is crucial to understand the strengths and limitations of humanized mice and select the most appropriate model. In this study, we describe the development of the human lymphoid and myeloid lineages using a flow cytometric analysis in four humanized mouse models derived from NOD mice xenotransplanted with CD34+ fetal cord blood from a single donor. Our results showed that all murine strains sustained human immune cells within a proinflammatory environment induced by GvHD. However, the Hu-SGM3 model consistently generated higher numbers of human T cells, monocytes, dendritic cells, mast cells, and megakaryocytes, and a low number of circulating platelets showing an activated profile when compared with the other murine strains. The hu-NOG-EXL model had a similar cell development profile but a higher number of circulating platelets with an inactivated state, and the hu-NSG and hu-NCG developed low frequencies of immune cells compared with the other models. Interestingly, only the hu-SGM3 and hu-EXL models developed mast cells. In conclusion, our findings highlight the importance of selecting the appropriate humanized mouse model for specific research questions, considering the strengths and limitations of each model and the immune cell populations of interest.

Immune-Mediated Pathogenesis in Dengue Virus Infection.

Dengue virus (DENV) infection is one of the major public health concerns around the globe, especially in the tropical regions of the world that contribute to 75% percent of dengue cases. While the majority of DENV infections are mild or asymptomatic, approximately 5% of the cases develop a severe form of the disease that is mainly attributed to sequential infection with different DENV serotypes. The severity of dengue depends on many immunopathogenic mechanisms involving both viral and host factors. Emerging evidence implicates an impaired immune response as contributing to disease progression and severity by restricting viral clearance and inducing severe inflammation, subsequently leading to dengue hemorrhagic fever and dengue shock syndrome. Moreover, the ability of DENV to infect a wide variety of immune cells, including monocytes, macrophages, dendritic cells, mast cells, and T and B cells, further dysregulates the antiviral functions of these cells, resulting in viral dissemination. Although several risk factors associated with disease progression have been proposed, gaps persist in the understanding of the disease pathogenesis and further investigations are warranted. In this review, we discuss known mechanisms of DENV-mediated immunopathogenesis and its association with disease progression and severity.

Humanized Mice for the Study of Dengue Disease Pathogenesis: Biological Assays.

Dengue is one of the most prevalent infectious diseases around the world, present in all continents and mainly affecting developing countries. With few tools to fight and study this disease, it is imperative to have reliable animal models that not only recapitulate human disease but also contain human components to understand the pathogenic mechanism and immune responses, allowing the development of new treatments and vaccines against dengue. Humanized mice are a significant advance in the development of in vivo models to understanding the relation of the human immune system and target organs such as the liver during the infection by dengue virus, allowing basic and preclinical research. In this chapter, we describe the use of humanized NSG mice (huNSG) for the study of dengue disease. The first model describes reconstitution of the human immune system by transplanting human CD34+ stem cells in newborn or adult NSG mice. The second model combines the reconstitution with CD34+ stem cells with the transplant of human primary hepatocytes. This dual reconstituted animal will have two of the major players involved in the development of dengue infection. However, there are still more biological components missing in this model for dengue, but researchers continue working to improve the huNSG model to reconstitute other human components.

Efficacy and safety of CD24Fc in hospitalised patients with COVID-19: a randomised, double-blind, placebo-controlled, phase 3 study.

BACKGROUND: Non-antiviral therapeutic options are required for the treatment of hospitalised patients with COVID-19. CD24Fc is an immunomodulator with potential to reduce the exaggerated inflammatory response to tissue injuries. We aimed to evaluate the safety and efficacy of CD24Fc in hospitalised adults with COVID-19 receiving oxygen support. METHODS: We conducted a randomised, double-blind, placebo-controlled, phase 3 study at nine medical centres in the USA. Hospitalised patients (age ≥18 years) with confirmed SARS-CoV-2 infection who were receiving oxygen support and standard of care were randomly assigned (1:1) by site-stratified block randomisation to receive a single intravenous infusion of CD24Fc 480 mg or placebo. The study funder, investigators, and patients were masked to treatment group assignment. The primary endpoint was time to clinical improvement over 28 days, defined as time that elapsed between a baseline National Institute of Allergy and Infectious Diseases ordinal scale score of 2-4 and reaching a score of 5 or higher or hospital discharge. The prespecified primary interim analysis was done when 146 participants reached the time to clinical improvement endpoint. Efficacy was assessed in the intention-to-treat population. Safety was assessed in the as-treated population. This study is registered with ClinicalTrials.gov, NCT04317040. FINDINGS: Between April 24 and Sept 22, 2020, 243 hospitalised patients were assessed for eligibility and 234 were enrolled and randomly assigned to receive CD24Fc (n=116) or placebo (n=118). The prespecified interim analysis was done when 146 participants reached the time to clinical improvement endpoint among 197 randomised participants. In the interim analysis, the 28-day clinical improvement rate was 82% (81 of 99) for CD24Fc versus 66% (65 of 98) for placebo; median time to clinical improvement was 6·0 days (95% CI 5·0-8·0) in the CD24Fc group versus 10·0 days (7·0-15·0) in the placebo group (hazard ratio [HR] 1·61, 95% CI 1·16-2·23; log-rank p=0·0028, which crossed the prespecified efficacy boundary [α=0·0147]). 37 participants were randomly assigned after the interim analysis data cutoff date; among the 234 randomised participants, median time to clinical improvement was 6·0 days (95% CI 5·0-9·0) in the CD24Fc group versus 10·5 days (7·0-15·0) in the placebo group (HR 1·40, 95% CI 1·02-1·92; log-rank p=0·037). The proportion of participants with disease progression within 28 days was 19% (22 of 116) in the CD24Fc group versus 31% (36 of 118) in the placebo group (HR 0·56, 95% CI 0·33-0·95; unadjusted p=0·031). The incidences of adverse events and serious adverse events were similar in both groups. No treatment-related adverse events were observed. INTERPRETATION: CD24Fc is generally well tolerated and accelerates clinical improvement of hospitalised patients with COVID-19 who are receiving oxygen support. These data suggest that targeting inflammation in response to tissue injuries might provide a therapeutic option for patients hospitalised with COVID-19. FUNDING: Merck & Co, National Cancer Institute, OncoImmune.

Campylobacter fetus aortitis in a patient with HIV.

A 36-year-old man with well controlled HIV developed Campylobacter fetus aortitis. To prevent aortic rupture, emergent surgical resection and neo-aortoiliac replacement with his left femoral vein was conducted. After surgical intervention, he was successfully treated with intravenous ertapenem for 6 weeks followed by oral amoxicillin for 3 months.

Current Trends and Limitations in Dengue Antiviral Research.

Dengue is the most prevalent arthropod-borne viral disease worldwide and affects approximately 2.5 billion people living in over 100 countries. Increasing geographic expansion of Aedes aegypti mosquitoes (which transmit the virus) has made dengue a global health concern. There are currently no approved antivirals available to treat dengue, and the only approved vaccine used in some countries is limited to seropositive patients. Treatment of dengue, therefore, remains largely supportive to date; hence, research efforts are being intensified for the development of antivirals. The nonstructural proteins, 3 and 5 (NS3 and NS5), have been the major targets for dengue antiviral development due to their indispensable enzymatic and biological functions in the viral replication process. NS5 is the largest and most conserved nonstructural protein encoded by flaviviruses. Its multifunctionality makes it an attractive target for antiviral development, but research efforts have, this far, not resulted in the successful development of an antiviral targeting NS5. Increase in structural insights into the dengue NS5 protein will accelerate drug discovery efforts focused on NS5 as an antiviral target. In this review, we will give an overview of the current state of therapeutic development, with a focus on NS5 as a therapeutic target against dengue.

Evolution of Nipah Virus Infection: Past, Present, and Future Considerations.

Nipah virus (NiV) is a zoonotic paramyxovirus of the Henipavirus genus first identified in Malaysia in 1998. Henipaviruses have bat reservoir hosts and have been isolated from fruit bats found across Oceania, Asia, and Africa. Bat-to-human transmission is thought to be the primary mode of human NiV infection, although multiple intermediate hosts are described. Human infections with NiV were originally described as a syndrome of fever and rapid neurological decline following contact with swine. More recent outbreaks describe a syndrome with prominent respiratory symptoms and human-to-human transmission. Nearly annual outbreaks have been described since 1998 with case fatality rates reaching greater than 90%. The ubiquitous nature of the reservoir host, increasing deforestation, multiple mode of transmission, high case fatality rate, and lack of effective therapy or vaccines make NiV's pandemic potential increasingly significant. Here we review the epidemiology and microbiology of NiV as well as the therapeutic agents and vaccines in development.

Safety and immunogenicity of an HIV-1 gp120-CD4 chimeric subunit vaccine in a phase 1a randomized controlled trial.

A major challenge for HIV vaccine development is to raise anti-envelope antibodies capable of recognizing and neutralizing diverse strains of HIV-1. Accordingly, a full length single chain (FLSC) of gp120-CD4 chimeric vaccine construct was designed to present a highly conserved CD4-induced (CD4i) HIV-1 envelope structure that elicits cross-reactive anti-envelope humoral responses and protective immunity in animal models of HIV infection. IHV01 is the FLSC formulated in aluminum phosphate adjuvant. We enrolled 65 healthy adult volunteers in this first-in-human phase 1a randomized, double-blind, placebo-controlled study with three dose-escalating cohorts (75 µg, 150 µg, and 300 µg doses). Intramuscular injections were given on weeks 0, 4, 8, and 24. Participants were followed for an additional 24 weeks after the last immunization. The overall incidence of adverse events (AEs) was not significantly different between vaccinees and controls. The majority (89%) of vaccine-related AE were mild. The most common vaccine-related adverse event was injection site pain. There were no vaccine-related serious AE, discontinuation due to AE, intercurrent HIV infection, or significant decreases in CD4 count. By the final vaccination, all vaccine recipients developed antibodies against IHV01 and demonstrated anti-CD4i epitope antibodies. The elicited antibodies reacted with CD4 non-liganded Env antigens from diverse HIV-1 strains. Antibody-dependent cell-mediated cytotoxicity against heterologous infected cells or gp120 bound to CD4+ cells was evident in all cohorts as were anti-gp120 T-cell responses. IHV01 vaccine was safe, well tolerated, and immunogenic at all doses tested. The vaccine raised broadly reactive humoral responses against conserved CD4i epitopes on gp120 that mediates antiviral functions.

Bench-to-bedside review: vaccine protection strategies during pandemic flu outbreaks.

Vaccination is the most effective means for the prevention of influenza, including pandemic strains. An ideal pandemic influenza vaccine should provide effective protection with the fewest number of doses in the shortest amount of time, and among the greatest proportion of the population. The current manufacturing processes required for embryonated chicken-egg-based influenza vaccines are limited in their ability to respond to pandemic situations - these limitations include problems with surge capacity, the need for egg-adapted strains, the possibility of contamination, and the presence of trace egg protein. Several vaccine strategies to circumvent the deficiencies intrinsic to an egg-based influenza vaccine are in various phases of development. These include the use of cell-culture-based growth systems, concomitant use of adjuvants, whole virus vaccines, recombinant protein vaccines, plasmid DNA vaccines, virus-like particle vaccines, and universal flu vaccines.