“Dengue fever overview and ongoing research of dengue vaccines and biologics.”

Global distribution of Dengue Fever Jan to Dec 2023 © ECDC [2005-2023].

FIGURE 1: Global distribution of Dengue Fever Jan to Dec 2023

“In 2023, over six million dengue cases and over 6000 dengue-related deaths were reported from 92 countries/territories (ECDC Jan 2024).” © ECDC [2005-2023].

Written by Michellie Hernandez, MD, with the help of ChatGPT

Published: August 08, 2024

Abstract: 

Dengue fever, the most common mosquito-borne viral infection, remains a significant public health concern, placing almost half the world at risk of contracting dengue fever (Akter et al., 2024). “In 2023, over six million dengue cases and over 6000 dengue-related deaths were reported from 92 countries/territories (Fig. 1 ECDC Jan 2024).” With its prevalence in tropical and subtropical regions, understanding the basics of Dengue is crucial for prevention and effective management. This paper provides an extensive overview of dengue and suggests an innovative approach to introducing the HOPE method to ongoing research for monoclonal antibodies (mAbs) specific against Dengue virus serotypes. As climate change increases the global population at risk of Dengue fever, there are continuing efforts for scalable and affordable solutions to reduce its morbidity and mortality rates by improving biologics like broadly neutralizing monoclonal antibodies specific against all DENV serotypes with a suggested method called the HOPE method.

INTRODUCTION:

“Each year, up to 400 million people are infected by a dengue virus. Approximately 100 million people get sick from infection, and 40,000 die from severe dengue (CDC June 2023),” Per WHO data, 2023 hit a near record high of new Dengue cases worldwide, 80% of which were reported in the Americas, affecting the Latin American communities in the Caribbean, Central America and South America the harshest. With global warming affecting the planet and spreading optimum conditions for the spread of mosquitoes, dengue is forming into an exponential global problem. With a 10-fold rise in dengue cases from 2000 till 2019, followed by a slight decline due to the COVID-19 pandemic, then a record high in 2023 with over 6 million dengue cases overtaking the second highest peak in 2019 of 5 million dengue cases (ECDC Jan 2024), WHO Global Situation website Dec 2023).

Pathogenesis of Dengue Fever:

Dengue fever is due to the infection of four Dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4 serotypes), which are part of the Flaviviridae family with positive sense RNA genome differentiated by their outer envelope proteins. In 2013, a new dengue virus submerged among primates in Malaysia, DENV-5, but the confirmation of DENV-5 as a causative agent of dengue fever in humans has not been determined (Akter et al., 2024). The dengue viruses infect humans primarily through the bite of infected female Aedes mosquitoes, specifically Aedes aegypti and Aedes albopictus mosquitoes. The optimal temperature for breeding Aedes mosquitoes is 25°C-30°C, as research data suggests that temperature determines the development and survival of these mosquitos (Liu et al., 2023). These mosquitoes are most active during the day, especially in the early morning and late afternoon. These mosquitoes are also responsible for other diseases as they are vectors for Zika, chikungunya, and other viruses. These four Dengue virus serotypes share similarities but have distinct surface antigens. Thus, infection with one serotype does not provide immunity to the others since the antibodies produced by one of the dengue serotypes would not recognize the surface antigen of another serotype. Therefore, individuals can be infected multiple times with different Dengue virus serotypes throughout their lifetime.

The dengue virus serotypes are spherical enveloped viruses containing a single strand of RNA consisting of 3 structural proteins (2 envelope proteins, E and M proteins, and one capsid protein, protein C) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Ormundo et al., 2023). The E proteins are superpositioned above the M proteins, forming the outer layer of the sphere. The M proteins undergo a maturation process that determines their level of the infectious state. “The second membrane protein (M) is used to aid in the folding, trafficking, and function of the fusion protein (E) (Zhang et al., 2013).” Upon replication of the dengue virus, a polyprotein containing the two envelope proteins is cleaved to a precursor of M protein (prM) and E protein within the endoplasmic reticulum. Together with the low pH and furin protease within the trans-Golgi apparatus, the prM is cleaved to its mature form (Zhang et al., 2013). 

The mature infectious dengue virus has a smooth outer icosahedral shell of 180 glycoproteins (E proteins) arranged in 90 dimers. Each E protein has three subunits called envelope dimer epitope (EDE), EDEI, EDEII, and EDE III, which all play a role in the ability of the virus to enter the cell. EDE II contains the fusion loop epitope (FLE). The mAbs specific against EDE and FLE are broadly neutralizing antibodies against all four virus serotypes. Specifically, the mAbs against EDE1 and EDE2 are the most potent and found to have less risk of complications from hemorrhagic fever (Dejnirattisai et al., 2015). 

These Dengue virus (DENV) serotypes replicate in the mosquito’s midgut and then are transported to the mosquito’s saliva. After a mosquito infected with the Dengue virus feasts on humans, the virus enters the bloodstream and replicates in various immune cells, including monocytes, macrophages, and dendritic cells. The infection of the immune cells leads to the release of cytokines, which are the mediators and activators of the immune system. These immune responses can contribute to the characteristic symptoms of Dengue fever. The incubation period between being bitten by an infected mosquito and the first signs of symptoms usually is around 8–12 days when the ambient temperature is between 25–28°C  (WHO March 2023).

Pathogenesis of Severe complications of dengue fever: Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS):

Dengue Hemorrhagic Fever (DHF) can progress to a shock called Dengue Shock Syndrome (DSS). Both can occur due to a combination of factors, including high viral load, secondary infection with a different Dengue virus serotype, and an overactive immune response. “Antibody-dependent enhancement (ADE) has been proposed to explain the increase in severity seen on the secondary infection (Dejnirattisai et al., 2015).” These factors can lead to a host immune T-cell response that triggers via cytokines widespread inflammation and damage to blood vessels, resulting in plasma leakage or vascular permeability, thrombocytopenia, and coagulopathy, causing hemorrhage (Halstead, 2007). All of these factors may lead to increased concentration of cells and solids in the blood due to the loss of plasma. Furthermore, it can lead to life-threatening shock if not promptly treated.

Symptoms of Dengue Fever:

Symptoms of Dengue Fever typically appear 4-10 days after being bitten by an infected mosquito. They may include (WHO March 2023):

• High fever

• Severe headache

• Pain behind the eyes

• Joint and muscle pain

• Nausea and vomiting

• Petechial Skin rash

• Signs and Symptoms of complications of Dengue Fever [Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS)] include severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, fatigue, and restlessness.

Diagnosis of Dengue Fever:

Diagnostic test results should be interpreted in conjunction with clinical evaluation, and the patient's symptoms and history of exposure to Dengue virus must be considered. Several diagnostic tests are available to identify Dengue viruses in individuals suspected of having Dengue fever. These tests can broadly be categorized into laboratory-based tests and point-of-care tests. Per the Centers for Disease Control and Prevention (CDC) review from 2019, “All patients with clinically suspected dengue should receive appropriate management to monitor for shock and reduce the risk of complications resulting from increased vascular permeability and plasma leakage and organ damage without waiting for diagnostic test results to be received (CDC June 2019).”

Here are some standard diagnostic methods: 

• “Dengue nonstructural protein 1 (NS1) antigen by immunoassay (CDC June 2019)”:

• “The non-structural dengue protein NS1 has been used as a biomarker in the diagnosis of dengue because it circulates widely in the blood during the acute phase of the infection and is associated with viremia [as well as a dengue hemorrhagic fever prognostic marker] (Chaturvedi et al., 2024).”

• Real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR): 

• rt-PCR are modified PCR designed to detect the presence of Dengue viral RNA in blood samples. “rt-PCR is becoming the “gold standard” of nucleic acid sequence detection and quantification [used for the detection, quantification, and genetic typing of microorganisms with a quick turnaround time of fewer than 24 hours] (Kaltenboeck et al., 2005).” This method can identify the virus and determine the specific serotype. Rt-PCR is a molecular technique that amplifies and detects highly sensitive and specific viral RNA or DNA in blood or tissue samples.

• If the serum sample is more than seven days after the onset of symptoms or if symptoms persist with a negative NAAT test run IgM Enzyme-Linked Immunosorbent Assay (IgM ELISA), IgM antibodies typically appear early in the course of infection. ELISA tests can help confirm Dengue infection and differentiate between primary (elevated IgM titers) and secondary infections (IgG titers will be elevated).

• Rapid Diagnostic Tests (RDTs) are convenient point-of-care tests that detect Dengue virus antigens or antibodies in blood samples. 

• These tests provide results within minutes (15 - 30 minutes) and can be helpful in resource-limited settings where laboratory infrastructure is lacking. However, RDTs may have lower sensitivity and specificity than PCR and ELISA tests. Therefore, the interpretation of rapid test results must be completed in conjunction with clinical evaluation, and the patient's symptoms and history of exposure to the Dengue virus must be considered.  Some common brand names of Dengue rapid tests include Dengue NS1 Ag Rapid Test, Dengue Duo Rapid Test, and SD Bioline Dengue Duo Rapid Test. “RDTs are qualitative tests that detect dengue NS1 antigen, IgM, and IgG to dengue virus based on the immunochromatographic method [visible to the naked eye] (Yow et al., 2021).”

• SD Bioline Dengue Duo: This test detects the Dengue virus NS1 antigen and Dengue-specific IgM/IgG antibodies in blood, serum, or plasma samples. It provides results within 15-30 minutes and is widely used to diagnose Dengue fever rapidly.

• Dengue NS1 Ag Rapid Test Kit: This test detects explicitly Dengue virus NS1 antigen in serum, plasma, or whole blood samples. It is designed for early detection of Dengue infection and provides results within 15-20 minutes.

• Panbio Dengue Early Rapid Test: This test detects Dengue virus NS1 antigen in serum, plasma, or whole blood samples. It is intended to diagnose Dengue infection early and can provide results within 15 minutes.

• Dengue IgM/IgG Rapid Test detects Dengue-specific IgM and IgG antibodies in serum, plasma, or whole blood samples. Dengue-specific IgM antibody detection indicates primary dengue infection, while Dengue-specific IgG antibody detection indicates a secondary dengue infection. This test result can be obtained within 15-20 minutes.

Diagnostic tests for Complications of Dengue Fever:

A Hemorrhagic Dengue Fever or Dengue shock syndrome (DSS) diagnosis must be made upon completion first of a clinical evaluation before any laboratory tests and imaging studies are indicated and interpreted. Early detection and treatment of any complication of dengue fever are essential for the patient’s prognosis.

Here are the main diagnostic tests used for DSS:

1. Clinical Evaluation should consist of an extensive assessment essential for diagnosing DSS. Healthcare providers evaluate the patient's medical history, symptoms, vital signs (blood pressure, heart rate, and temperature), and physical examination findings. Clinical signs of DSS may include rapid pulse, weak peripheral pulses, cold, clammy skin, decreased urine output, and signs of shock (e.g., altered mental status, hypotension).

2. Laboratory Tests: Several laboratory tests can help diagnose DSS, including Complete blood count, which helps assess platelet levels, hematocrit levels, and white blood cell count.

3. Coagulation profile: This test evaluates clotting factors and platelet function to check for abnormal bleeding or clotting. This profile includes prothrombin time (PT), activated partial thromboplastin time (aPTT), and levels of fibrinogen and fibrin degradation products, which can help assess for coagulopathy, which may occur in severe Dengue cases.

4. Liver function tests, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), assess liver enzymes and function, as liver involvement is often detected in severe dengue cases.

5. Imaging Studies: In cases of severe abdominal pain or suspected internal bleeding due to DSS, imaging studies such as ultrasound or computed tomography scans may be used to evaluate the extent of organ damage, particularly in the liver or spleen. These tests may be performed to assess for complications such as ascites, pleural effusion, or bleeding.

6. Hemodynamic Monitoring: Continuous monitoring of hemodynamic parameters, including blood pressure, heart rate, central venous pressure, and urine output, is crucial for assessing the fluid status and guiding fluid resuscitation in patients with DSS.

Treatment of Dengue Fever:

There is no specific FDA-approved antiviral treatment for Dengue fever as of yet. However, ongoing human clinical trials evaluate the safety and efficacy of neutralizing monoclonal antibodies specific against all four dengue viruses and other potential treatments that have shown potential in preclinical research. Currently, FDA-approved treatments primarily focus on relieving symptoms and providing supportive care (CDC September 2021).

Current FDA-approved treatment for dengue may include:

• Fluid Replacement: Drinking plenty of fluids to prevent dehydration is crucial, especially if experiencing vomiting or high fever. In severe cases, intravenous fluids may be necessary.

• Acetaminophen (paracetamol) is the currently recommended drug of choice to help reduce fever and relieve pain since aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) like ibuprofen can increase the risk of bleeding.

• Rest is essential for recovery from Dengue fever.

• Severe cases of Dengue hemorrhagic fever or Dengue shock syndrome require hospitalization for close monitoring and supportive care, such as blood transfusions or intravenous fluids.

Current Prevention Measures of Dengue Fever:

Since there is currently no specific antiviral treatment for Dengue fever, prevention strategies play are essential in controlling the propagation of the disease. After all, controlling the mosquito population will reduce the risk of dengue fever among the population. Some prevention strategies for Dengue fever include:

• Eliminate stagnant water sources where mosquitoes can breed, such as empty containers, flower pots, and tires. 

• Use insect repellent containing DEET on exposed skin and clothing.

• Reduce the exposed skin area by wearing long-sleeved shirts and long pants where dengue is endemic.

• Prevent the entry of mosquitos indoors by using screened doors and windows.

• Use mosquito nets while sleeping during the early hours of the morning.

• Public health should consider the implementation of community-wide fumigation efforts or vector control measures.

Vaccines:

As of 2023, two live attenuated Dengue vaccines, Dengavaxia and Qdenga, have been developed. However, since they are both considered altered live viruses, they are not considered adequate for pregnant or immunocompromised individuals. 

Sanofi Pasteur developed the first licensed dengue vaccine in 2015, Dengvaxia (CYD-TDV), a live-attenuated recombinant vaccine that offers 60% protection against all four Dengue virus serotypes (tetravalent). However, its indication is now only within endemic areas, as its efficiency data on a third-year follow-up study indicated a decrease in efficiency in individuals who had no prior dengue exposure, especially in children under nine years old (Angelin et al., 2023). As of 2024, Dengvaxia is licensed for use in the US, EU, Asian, and Latin American countries (Sanofi April 2024). Dengvaxia’s efficacy varies by serotype, with less efficacy in DENV-1 and DENV-2 infections. The manufacturing license of Sanofi Pasteur Dengvaxia was granted based on the vaccine development research done at Washington and St. Louis Universities that excised the prM and E protein genes from a yellow fever virus and substituted them with the corresponding prM and E proteins of each of the dengue virus serotypes.

“Denvax (TAK-003) by Takeda is a live attenuated tetravalent dengue vaccine initially developed by the Division of Vector-Borne Diseases of the Centers for Disease Control and Prevention (CDC). TAK-003 contains a DENV-2 backbone whereby the pre-membrane (prM) and envelope (E) structural genes are substituted with the chimeric viruses for DENV1, DENV3, and DENV4 (Kariyawasam et al., 2023).” Denvax (TAK-003), also known as Qdenga commercially is currently licensed in Europe, the U.K., Brazil, Argentina, Indonesia, and Thailand for use on individuals four years and older, with restrictions among pregnant or immunocompromised individuals (Takeda, July 2023). Per one-year follow-up studies after the Qdenga vaccination, the average efficiency was 80% against all four Dengue virus serotypes, varying among serotypes. The highest efficiency was against DENV2 (98%), and the lowest efficiency was in DENV3 (63%) (Angelin et al., 2023). The efficacy of Qdenga reduced on average to 59% over time per four-year follow-up studies, indicating the need for booster shots, especially among vaccinated individuals with no prior exposure since data from these studies showed reduced efficacy against DENV3 and DENV4 among these individuals.  

Challenges in Dengue Vaccine Development:

Challenges in Dengue vaccine development include achieving broad and long-lasting immunity and addressing vaccine safety concerns. The DENV multiple serotypes and Antibody-Dependent Enhancement (ADE) leading to the complications of dengue fever (DHF and HSS) require a proper balancing act in the immune response with the assurance that neutralizing antibodies are developed and efficient on all serotypes for long-lasting immunity. Due to the global scale of people at risk and the potential need for booster shots, the scalability of vaccine production and the affordability of vaccines for the population at risk is a significant challenge. Ongoing research is being done to address these challenges; several vaccine development companies are using recombinant DNA technology to scale up the production of vaccines in a cost-effective manner. Several other dengue vaccines are in preclinical animal studies with promising data for clinical trials.

Monoclonal antibodies (mAbs) specific against dengue viruses:

The development of monoclonal antibodies (mAbs) for diagnosing and treating dengue virus infections represents a significant scientific endeavor to improve public health outcomes worldwide. “Monoclonal antibodies could function as prophylaxis (i.e., for the prevention of malaria), or could be used to treat (tropical) infections (i.e., rabies, dengue fever, yellow fever) (de Jong et al., 2024).” As of 2023, the only FDA-approved mAbs specific against infectious viral pathogens have been only three viruses: respiratory syncytial virus (RSV), human immunodeficiency virus (HIV-1), and Ebola virus (EBOV). The COVID-19 pandemic made an exception with SARSCOV-2, which was granted FDA emergency use authorization due to the need to speed up the R&D process of mAbs development during the pandemic. The discovery process of identifying neutralizing mAbs within human individuals and animal models infected with dengue has advanced with the help of modern techniques isolating B cells from the serum with fluorescence-activated cell sorting (FACS) and microfluidic platforms like Berkeley Lights BeaconTM. Dengue-neutralizing antibodies have been found in infected individuals to be specific against E, prM, and NS1 proteins (Ormundo et al., 2023).

Key advancements in the development of biologics specific against dengue include:

• Broadly Neutralizing Antibodies: Studies have shown that some mAbs can neutralize multiple dengue virus serotypes by targeting conserved regions within the envelope protein. These discoveries are crucial for developing effective treatments against all dengue virus serotypes (Dejnirattisai et al., 2015). The most advanced neutralizing mAb specific against all 4 Dengue virus serotypes in the R&D process is a recombinant mAb (VIS513). Similar mAbs against dengue viruses AV-1 and Dengushield are in phase 1 clinical trials.

• VIS513 (Visterra Biotechnology, Cambridge, MA, USA), currently in phase 2 clinical trials (CTRI/2021/07/035290). VIS513 is a genetically engineered humanized IgG1 neutralizing antibody specific against all four serotypes of DENV, specifically the E protein domain III (EDIII) (Robinson et al., 2015).

• AV-1 (AbViro LLC, Bethesda, MD, USA) is currently in phase 1 clinical trials (NCT04273217). Details on the preclinical data results and target have yet to be published.

• Dengushield (Serum Institute of India, Pune, India) combined efforts with Visterra to continue its research with VIS513 and recently published its phase 1 clinical trial (NCT03883620) results passing the safety protocols of phase 1 clinical trials (Gunale et al., 2024). Continuation of phase 2 clinical trials is still pending.

• Enhancement of Antibody Potency and Breadth: Through techniques such as antibody engineering and affinity maturation, researchers have enhanced the potency and breadth of mAbs neutralization against the dengue virus. One antibody engineering technique includes modifying the Fc regions of antibodies to improve their interaction with immune cells. Animal studies have indicated the role of Fc areas in the therapeutic efficacy of mAbs in preventing complications of dengue viruses. “Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV (Tien et al., 2022).”

• Therapeutic Efficacy in Preclinical Models: A few mAbs have progressed to clinical trials, demonstrating efficacy in animal models. The preclinical studies with animal models have provided foundational data supporting the potential of mAbs in treating or preventing severe dengue disease manifestations.

Challenges in Monoclonal Antibody Development:

Despite these advances, there are significant challenges:

• Complexity of Dengue Virus Immunity: The antibody-dependent enhancement (ADE) phenomenon is a significant hurdle. ADE occurs when non-neutralizing or sub-neutralizing antibodies from an initial dengue infection facilitate virus entry of a second dengue infection into host cells, potentially leading to severe disease. The ADE phenomenon complicates the development of mAbs because antibodies effective against one serotype might exacerbate infection when exposed to another serotype if not shown to be a potent enough neutralizing antibody within the individual. Thus, the ideal therapeutic mAbs specific against dengue viruses must be a broadly neutralizing antibody against all four dengue viruses since these mAbs are specific against constant regions of DENV that tend not to mutate, thus less risk of loss of efficacy of the mAbs over time. 

• Serotype Specificity and Cross-Reactivity: Developing mAbs that are equally effective against all four serotypes without causing ADE remains challenging. The variability across serotypes can lead to differential binding and neutralization efficiency, which must be carefully balanced in therapeutic applications.

• Scalability and Accessibility: Since half the world’s population is at risk of dengue virus infection, producing mAbs at scale and ensuring they are accessible to populations in dengue-endemic regions are also significant challenges. Monoclonal antibodies are generally expensive to produce, which could limit their availability in lower-income countries. Although recombinant mAbs with genetic engineering and DNA technology have reduced the cost of mAbs for lab and diagnostic purposes, FDA  acceptance of the recombinant mAbs as a therapeutic is still challenging.

DISCUSSION: 

Another approach to facing the challenges of dengue fever is to rapidly improve research that improves the biologics, specifically broadly neutralizing monoclonal antibodies specific against all DENV serotypes. Concentrating on mAbs production rather than vaccine production might reduce the population and supply chain demand, reducing the scale and cost of R&D production. Instead of administering vaccines to the population at risk, which is half the global population, pharmaceutical companies can concentrate on the production of mAbs for confirmed dengue cases, which ranges from 100 to 400 million yearly, to reduce the complications of dengue thus, the mortality rate due to dengue.

In 2020, I (Dr. Hernandez) created and publicly suggested the HOPE method for the R&D process for mAbs and bio proteins. Focusing on isolating antibodies of recovered patients since isolating B cells from serum might limit the actual variation of antibodies towards an antigen of interest that can be detected in the serum. The HOPE method uses computational models to reverse engineer the antibodies without knowing the genes found in B cells (Hernandez et al., 2022). I also suggested using recombinant DNA technology from the genes obtained from the isolation of B cells to reduce the cost of mAbs production. I recommend applying the HOPE method to obtain mAbs specific against the dengue serotypes, which will consist of more studies collecting serum samples from recovered humane patients to detect broadly neutralizing antibodies whose FAB region can be reverse-engineered toward mAbs production specific against all the dengue virus serotypes. Fc region analysis can also help enhance future mAbs development. These studies identifying the broadly neutralizing antibodies in recovered patients might also help identify new epitopes of interest that these antibodies bind to advance vaccine production. Ongoing research in Exscientia AI is developing a de novo protein design similar to the machine learning models suggested by the HOPE method to reverse engineering antibodies. Exscientia AI models predict the inverse folding of antibody proteins to their linear AA sequence to advance drug discovery (Dreyer et al., 2023). Hopefully, Exscientia AI models, together with the rest of the steps of the HOPE method, will shed light on new developments for future diagnostic and therapeutic mAbs development for dengue and many other diseases.

The HOPE Method applied to Dengue Viral Infections:

The HOPE Method by Dr. Michellie Hernandez edited illustration of Deb Bose (Hernandez et al., 2023)

Figure 2

Developing mAbs specific against Dengue serotypes with the HOPE Method: 

(1) Research antibodies from recovered Dengue patient populations to select the best antibody to reverse engineer. 

(2) Reverse engineering the Fab and Fc regions of the selected antibody to its linear amino acid sequence with de novo protein sequencing and computational models. 

(3) Reverse engineering the Fab and Fc region from its linear amino acid sequence to its codon sequence, followed by its mRNA sequence, using reverse translation and codon engineering computational models. 

(4) Union of the mRNA sequence of both Fab and Fc regions with genetic engineering. 

(5 & 6) Create in vitro transcribed mRNA (IVT-mRNA) without nanoparticles and its decoded DNA and place it within a plasmid to induce the mass production of HOPE mAb via recombinant DNA technology in yeast or larger plant species.

The HOPE Method is a six-step process of mAbs production from antibodies collected from blood samples of human donors with a potent adaptive immune response against a pathogen of interest (Hernandez et al., 2023).

1. Obtain blood samples from recovered patient populations with confirmed cases of dengue infection to identify broadly neutralizing antibodies against all known DENV serotypes. The isolated antibodies should be compared in efficacy and safety to mAbs currently in clinical trials like VIS315 and Dengushield or with a history of efficacy in animal studies like NS1 or other EDIII-specific mAbs. This step is a series of studies to obtain the optimal antibody showing efficacy against all serotypes within the 90% range or above.

2. HPLC mass spectrometer and cryogenic electron microscopy (cryo-EM) were used to select and analyze the best antibody identified in Step 1. This data can be entered into trained computational models to complete de novo peptide sequencing of the antibody's linear amino acid sequence, accomplishing further decoding of the mRNA sequence of the selected antibody’s FAB (the antibody's binding site to the antigen) and FC region in Step 3.

3. Decode the mRNA sequence of the FAB and Fc region from the predicted linear amino acid sequence in the computational models in Step 2 using reverse translation and codon engineering computational models. In natural translation, the mRNA sequence codes for an amino acid with three nucleic acids combined called the codon. In reverse translation or back translation on some articles, the mRNA is predicted from linear amino acid sequence data based on algorithms from trained computational models that have analyzed the codon chart and numerous natural translations of mRNA sequence to its known amino acid sequences, thus creating an algorithm to decode mRNA from linear amino acid sequences.

4. Unite the mRNA sequences of the FAB and FC region obtained in step 3 so that the union of the two encodes a complete, fully human monoclonal antibody (mab) effective against all DENV serotypes.

5. Obtain an In Vitro transcribed mRNA (IVT mRNA) encoding the combined mRNA sequence (Hernandez et al., 2023).

6. “Synthesize a synthetic DNA sequence that, upon transcription, will transcribe the mRNA sequence in Step 5 (IVT mRNA without the delivery system) via reverse transcription and genetic engineering. The synthetic DNA is inserted into a plasmid, and with the use of recombinant DNA technology in E. Coli or yeast culture (Ridder et al., 1995), clones of IVT mRNA could be reproduced (Hernandez et al., 2023).”

CONCLUSION

In conclusion, the global impact of dengue fever is a significant public health concern, with over six million cases reported in 2023 and a high mortality rate. The main challenges in dengue vaccine development are achieving broad and long-lasting immunity, addressing vaccine safety concerns, and scalability of vaccine production, which are crucial areas of ongoing research. In animal studies, monoclonal antibodies (mAbs) have shown promise in treating and preventing severe dengue disease manifestations. However, there are challenges in transitioning towards clinical applications regarding their development, production, and accessibility. The challenges in mAb development, such as the complexity of dengue virus immunity, serotype specificity, cross-reactivity, scalability, and accessibility, can potentially be addressed by the HOPE method, which involves reverse engineering antibodies of recovered patients and bioproteins, as a potential approach for mAb development. By highlighting ongoing research efforts, including advancements in broadly neutralizing antibodies and the use of modern techniques for antibody discovery and development of broadly neutralizing mAb against all of the DENV serotypes, yearly mortality rates can decrease as prevention of the complications of dengue fever is addressed. The potential of artificial intelligence (AI) models to advance drug discovery might shed light on new developments for future diagnostic and therapeutic mAbs development for dengue and other diseases. The HOPE method is a suggested proposal to improve ongoing research in mAbs production, vaccine strategies, and future aspects of dengue fever.

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Acknowledgments: 

The author would like to thank all the scientists who have dedicated their efforts to tropical medicine.

Funding: Independent funding was provided by the MD Biomimicry pro bono project on Dengue Fever.

Author contributions: Dr. Michellie Hernandez (MH) and Artificial Intelligence

Conceptualization: MH, ChatGPT, and PopAI

Methodology: MH

Investigation: MH

Visualization: MH

Funding acquisition: MH

Project administration: MH

Supervision: MH

Writing – original draft: MH, ChatGPT, and PopAI

Writing – review & editing: MH and Grammarly

Competing interests: The author denies any competing interests.