Patent Grant
11667697
The present disclosure relates generally to the fields of medicine, infectious disease, and immunology. More particular, the disclosure relates to human antibodies binding to orthopoxvirus.
Naturally-occurring members of the Orthopoxvirus genus, cowpox virus (CPXV), monkeypox virus (MPXV) and Variola virus (VARV), cause severe infections in humans. VARV exclusively causes human infections, with an estimated 300-500 million deaths during the 20th century before the initiation of the global smallpox vaccination campaign (Smith and McFadden, 2002). MPXV and CPXV are emerging zoonotic infections with a sporadic occurrence worldwide (McCollum et al., 2015; Reed et al., 2004; Vorou et al., 2008). There is no licensed specific treatment for these infections, and the only method of prevention is vaccination using vaccinia virus (VACV). Vaccinations against smallpox were discontinued in the late 1970s, leaving a large proportion of the current human population vulnerable to orthopoxviruses. The fear that smallpox could potentially re-emerge following a bioterror or biowarfare action (Smith and McFadden, 2002), the sporadic outbreaks of zoonotic MPXV and CPXV, and the increasing prevalence of immunocompromised individuals who cannot be vaccinated safely (Kemper et al., 2002), has stimulated renewed interest in research on orthopoxvirus protective immunity and treatment.
Poxviruses have a large and complex proteome containing over 200 proteins. During infection, the virus exists in two antigenically distinct forms, designated mature virions (MV) or enveloped virions (EV), which contain ˜25 or 6 surface proteins, respectively (Moss, 2011). Monkeypox and smallpox are select agents and subject to the select agent regulations under (42 C.F.R. § 73). Various poxvirus species share many genetic and antigenic features (Hughes et al., 2010; Ichihashi and Oie, 1988; Stanford et al., 2007), and an infection with an orthopoxvirus of any one species may confer substantial protection against infection with the other orthopoxviruses (McConnell et al., 1964). Vaccination with VACV protects against disease caused by VARV, MPXV, or CPXV (Hammarlund et al., 2005). The immunologic mechanisms underlying cross-protection by immunization with VACV likely are diverse, but include neutralizing antibodies (Moss, 2011). A critical role for antibodies (Abs) in poxvirus immunity was suggested by historical cases in which passive transfer of serum from VARV- or VACV-immune subjects protected exposed individuals against smallpox (Kempe et al., 1961). Recent studies in non-human primate or murine models of experimental infection showed that polyclonal Abs are necessary and sufficient for protection against lethal challenge with MPXV or VACV (Belyakov et al., 2003; Edghill-Smith et al., 2005). The level of neutralizing activity in immune serum is thought to be the best laboratory predictor of protective immunity to orthopoxvirus infections in humans (Mack et al., 1972). Human vaccinia immune globulin (VIG) has been used for the prevention and treatment of some smallpox and vaccine-related complications with limited success (Wittek, 2006). The level of efficacy is uncertain due to lot-to-lot variation in potency and a lack of understanding of the molecular determinants of protection.
Percutaneous inoculation with VACV elicits a broad and heterogeneous serum Ab response that targets a large number of antigenic determinants of VACV (Davies et al., 2005a; Davies et al., 2007). The viral inhibitory activity of serum from immune subjects with cross-neutralizing activity to VACV, MPXV, and VARV likely is composed of Abs to diverse specificities (Hughes et al., 2012; Kennedy et al., 2011). Abs in VIG recognize many antigen targets, including surface proteins of both EV and MV virion forms of VACV (Davies et al., 2005a). Study of polyclonal Abs in poxvirus-immune sera of rabbits revealed the pattern of recognition for each poxvirus was unique, but also suggested that different poxvirus species shared common neutralizing determinants (Baxby, 1982). Studies in murine infection models identified targets for neutralizing and protective mouse monoclonal Abs (mAbs), which included the MV surface proteins A27, L1, H3, D8, A28, A13 and A17, and the EV surface proteins B5 and A33 (Moss, 2011). Protection of mice against systemic and respiratory infection with murine Abs required clones specific to antigens of both MV and EV forms of VACV (Lustig et al., 2005). These studies suggest complex patterns of recognition by Abs protecting against infection and disease in experimental animal models, but the molecular basis for neutralization and cross-reactive poxvirus immunity in humans are poorly understood.
Thus, in accordance with the present disclosure, there is provided a method of detecting a orthopoxvirus infection in a subject comprising (a) contacting a sample from said subject with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) detecting orthopoxvirus in said sample by binding of said antibody or antibody fragment to a orthopoxvirus antigen in said sample. The sample may be a body fluid, and may be blood, sputum, tears, saliva, mucous or serum, urine, exudate, transudate, tissue scrapings or feces. Detection may comprise ELISA, RIA or Western blot. The antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The method may further comprise performing steps (a) and (b) a second time and determining a change in orthopoxvirus antigen levels as compared to the first assay.
The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.
In another embodiment, there is provided a method of treating a subject infected with orthopoxvirus, or reducing the likelihood of infection of a subject at risk of contracting orthopoxvirus, comprising delivering to the subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The antibody may be an IgG. The antibody may be is a chimeric antibody. The antibody or antibody fragment may be administered prior to infection, or may be administered after infection. Delivering may comprise antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.
The antibody or antibody fragment may be encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1, may be encoded by clone-paired light and heavy chain variable sequences having 95% identify to as set forth in Table 1, and may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired sequences from Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.
Also provided is a monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The antibody may be a chimeric antibody, or is bispecific antibody. The antibody may be an IgG. The antibody or antibody fragment may further comprise a cell penetrating peptide and/or is an intrabody.
The antibody or antibody fragment may be encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1, may be encoded by clone-paired light and heavy chain variable sequences having 95% identify to as set forth in Table 1, and may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired sequences from Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.
In yet another embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The antibody may be a chimeric antibody or a bispecific antibody. The antibody may be an IgG. The antibody or antibody fragment may further comprise a cell penetrating peptide and/or is an intrabody.
The antibody or antibody fragment may be encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1, may be encoded by clone-paired light and heavy chain variable sequences having 95% identify to as set forth in Table 1, and may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired sequences from Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.
A further embodiment comprises a vaccine formulation comprising two or more antibodies or antibody fragments characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. At least one of said two or more antibody fragments may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. At least one of said antibodies may be a chimeric antibody, or is bispecific antibody. At least one of said antibodies may be an IgG. At least one of said antibodies or antibody fragments further comprises a cell penetrating peptide and/or is an intrabody.
At least one of the two or more antibodies or antibody fragments may be encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1, may be encoded by clone-paired light and heavy chain variable sequences having 95% identify to as set forth in Table 1, and may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired sequences from Table 1. At least one of the two or more antibodies or antibody fragments may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.
The vaccine formulation may comprise antibodies or antibody fragments that bind to MV and EV forms of vaccinia virus, such as where formulation comprises at least two antibodies or antibody fragments that bind to each of MV and EV forms of vaccinia virus. The vaccine formulation may comprise antibodies that bind to two or more of the orthopox antigens selected from the group consisting of A27, D8, L1, B5, A33 and H3, such as wherein said formulation may comprise antibodies or antibody fragments that bind to MV proteins A27 and L1 and EV proteins B5 and A33, and may further comprise antibodies or antibody fragments that bind to MV proteins D8 and H3.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
As discussed above, immunization with vaccinia virus (VACV) induces long lasting cross-protective immunity to Variola virus (VARV) and other clinically important orthopoxviruses, such as cowpox virus (CPXV) and monkeypox virus (MPXV). The appearance of serum neutralizing antibodies (Abs) induced by VACV may be a correlate of immunity for orthopoxviruses. However, the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses in humans remains unknown. The inventors generated a large panel of orthopoxvirus-specific human monoclonal Abs from VACV-immunized subjects or from a subject with history of naturally-acquired MPXV infection. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV and VARV and that are determinants of protection in murine challenge models. Optimal protection against infection and disease following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus, and the presence of complement. This work reveals the principal targets for human Abs that mediate cross-protective immunity to diverse orthopoxviruses, using complementary and cooperative neutralizing activities. These and other aspects of the disclosure are described in detail below.
Four genera of poxviruses may infect humans: orthopoxvirus, parapoxvirus, yatapoxvirus, and molluscipoxvirus. Orthopox include smallpox virus (Variola), vaccinia virus, cowpox virus, and monkeypox virus. Parapox include orf virus, pseudocowpox, and bovine papular stomatitis virus. Yatapox include tanapox virus and yaba monkey tumor virus; Molluscipox include molluscum contagiosum virus (MCV). The most common are vaccinia (seen on Indian subcontinent) and molluscum contagiosum, but monkeypox infections are rising (seen in west and central African rainforest countries). Smallpox has largely been eradicated by vaccination, but concerns remain in light of the growing unvaccinated population.
Poxviridae viral particles (virions) are generally enveloped (external enveloped virion-EEV), though the intracellular mature virion (IMV) form of the virus, which contains different envelope, is also infectious. They vary in their shape depending upon the species but are generally shaped like a brick or as an oval form similar to a rounded brick because they are wrapped by the endoplasmic reticulum. The virion is exceptionally large, its size is around 200 nm in diameter and 300 nm in length and carries its genome in a single, linear, double-stranded segment of DNA. By comparison, Rhinovirus is 1/10 as large as a typical Poxviridae virion.
Replication of the poxvirus involves several stages. The first thing the virus does is to bind to a receptor on the host cell surface; the receptors for the poxvirus are thought to be glycosaminoglycans (GAGs). After binding to the receptor, the virus enters the cell where it uncoats. Uncoating of the virus is a two-step process. Firstly the outer membrane is removed as the particle enters the cell; secondly the virus particle (without the outer membrane) fuses with the cellular membrane to release the core into the cytoplasm. The pox viral genes are expressed in two phases. The early genes encode the non-structural protein, including proteins necessary for replication of the viral genome, and are expressed before the genome is replicated. The late genes are expressed after the genome has been replicated and encode the structural proteins to make the virus particle. The assembly of the virus particle occurs in five stages of maturation that lead to the final exocytosis of the new enveloped virion. After the genome has been replicated, the immature virion (IV) assembles the A5 protein to create the intracellular mature virion (IMV). The protein aligns and the brick-shaped envelope of the intracellular enveloped virion (IEV). These IEV particles are then fused to the cell plasma to form the cell-associated enveloped virion (CEV). Finally, the CEV encounters the microtubules and the virion prepares to exit the cell as an extracellular enveloped virion (EEV). The assembly of the virus particle occurs in the cytoplasm of the cell and is a complex process that is currently being researched to understand each stage in more depth. Considering the fact that this virus is large and complex, replication is relatively quick taking approximately 12 hours until the host cell dies by the release of viruses.
The replication of poxvirus is unusual for a virus with double-stranded DNA genome (dsDNA) because it occurs in the cytoplasm, although this is typical of other large DNA viruses. Poxvirus encodes its own machinery for genome transcription, a DNA dependent RNA polymerase, which makes replication in the cytoplasm possible. Most dsDNA viruses require the host cell's DNA-dependent RNA polymerase to perform transcription. These host DNA are found in the nucleus, and therefore most dsDNA viruses carry out a part of their infection cycle within the host cell's nucleus.
The ancestor of the poxviruses is not known but structural studies suggest it may have been an adenovirus or a species related to both the poxviruses and the adenoviruses. Based on the genome organisation and DNA replication mechanism it seems that phylogenetic relationships may exist between the rudiviruses (Rudiviridae) and the large eukaryal DNA viruses: the African swine fever virus (Asfarviridae), Chlorella viruses (Phycodnaviridae) and poxviruses (Poxviridae). The mutation rate in these genomes has been estimated to be 0.9-1.2×10−6 substitutions per site per year. A second estimate puts this rate at 0.5-7×10−6 nucleotide substitutions per site per year. A third estimate places the rate at 4-6×10−6.
The last common ancestor of the extant poxviruses that infect vertebrates existed 0.5 million years ago. The genus Avipoxvirus diverged from the ancestor 249±69 thousand years ago. The ancestor of the genus Orthopoxvirus was next to diverge from the other clades at 0.3 million years ago. A second estimate of this divergence time places this event at 166,000±43,000 years ago. The division of the Orthopox into the extant genera occurred 14,000 years ago. The genus Leporipoxvirus diverged 137,000±35,000 years ago. This was followed by the ancestor of the genus Yatapoxvirus. The last common ancestor of the Capripoxvirus and Suipoxvirus diverged 111,000±29,000 years ago. An isolate from a fish—Salmon Gill Poxvirus—appears to be the earliest branch in the Chordopoxvirinae.
A. Taxonomy
The name of the family, Poxviridae, is a legacy of the original grouping of viruses associated with diseases that produced poxes in the skin. Modern viral classification is based on phenotypic characteristics; morphology, nucleic acid type, mode of replication, host organisms, and the type of disease they cause. The smallpox virus remains as the most notable member of the family.
The species in the subfamily Chordopoxvirinae infect vertebrates and those in the subfamily Entomopoxvirinae infect insects. There are 10 recognized genera in the Chordopoxvirinae and 3 in the Entomopoxvirinae. Both subfamilies also contain a number of unclassified species for which new genera may be created in the future. Cotia virus is an unusual virus that may belong to a new genus.
The GC-content of these genomes differs considerably. Avipoxvirus, Capripoxvirus, Cervidpoxvirus, Orthopoxvirus, Suipoxvirus, Yatapoxvirus and one Entomopox genus (Betaentomopoxvirus) along with several other unclassified Entomopoxviruses have a low G+C content while others—Molluscipoxvirus, Orthopoxvirus, Parapoxvirus and some unclassified Chordopoxvirus—have a relatively high G+C content. The reasons for these differences are not known.
Phylogenetic analysis of 26 Chordopoxviruses genomes has shown that the central region of the genome is conserved and contains ˜90 genes. The termini in contrast are not conserved between species. Of this group Avipoxvirus is the most divergent. The next most divergent is Molluscipoxvirus. Capripoxvirus, Leporipoxvirus, Suipoxvirus and Yatapoxvirus genera cluster together: Capripoxvirus and Suipoxvirus share a common ancestor and are distinct from the genus Orthopoxvirus. Within the Othopoxvirus genus Cowpox virus strain Brighton Red, Ectromelia virus and Monkeypox virus do not group closely with any other member. Variola virus and Camelpox virus form a subgroup. Vaccinia virus is most closely related to CPV-GRI-90.
B. Vaccinia Virus
The prototypial poxvirus is vaccinia virus, known for its role as the active agent in the eradication of smallpox. The vaccinia virus is an effective tool for foreign protein expression, as it elicits a strong host immune-response. The vaccinia virus enters cells primarily by cell fusion, although currently the receptor responsible is unknown. Vaccinia virus is closely related to the virus that causes cowpox; historically the two were often considered to be one and the same. The precise origin of vaccinia virus is unknown due to the lack of record-keeping as the virus was repeatedly cultivated and passaged in research laboratories for many decades. The most common notion is that vaccinia virus, cowpox virus, and Variola virus (the causative agent of smallpox) were all derived from a common ancestral virus. There is also speculation that vaccinia virus was originally isolated from horses.
In addition to the morbidity of uncomplicated primary vaccination, transfer of infection to other sites by scratching, and post vaccinial encephalitis, other complications of vaccinia infections may be divided into the following types: Generalized vaccinia, Eczema vaccinatum, Progressive vaccinia (Vaccinia gangrenosum, Vaccinia necrosum) and Roseola vaccinia.
Vaccinia contains three classes of genes: early, intermediate and late. These genes are transcribed by viral RNA polymerase and associated transcription factors. Vaccinia replicates its genome in the cytoplasm of infected cells, and after late-stage gene expression undergoes virion morphogenesis, which produces IMV contained within an envelope membrane. The exact origin of the envelope membrane is still unknown. The IMV is then transported to the Golgi apparatus where it is wrapped with an additional two membranes, becoming the Intracellular Enveloped Virus (IEV). The IEV is transported along cytoskeletal microtubules to reach the cell periphery, where it fuses with the plasma membrane to become the Cell-associated Enveloped Virus (CEV). This triggers actin tails on cell surfaces or is released as EEV.
Vaccinia virus is able to undergo multiplicity reactivation. MR is the process by which two, or more, virus genomes containing otherwise lethal damage interact within an infected cell to form a viable virus genome. Abel found that vaccinia viruses exposed to doses of UV light sufficient to prevent progeny formation when single virus particles infected host chick embryo cells, could still produce viable progeny viruses when host cells were infected by two or more of these inactivated viruses; that is, MR could occur. Researchers have demonstrated MR of vaccinia virus after treatment with UV-light, nitrogen mustard, and X-rays or gamma rays.
Vaccinia contains within its genome several proteins that give the virus resistance to interferons. K3L is a protein with homology to the protein eukaryotic initiation factor 2 (eIF-2alpha). K3L protein inhibits the action of PKR, an activator of interferons. E3L is another protein encoded by Vaccinia. E3L also inhibits PKR activation; and is also able to bind to double stranded RNA.
C. Smallpox
Smallpox was an infectious disease caused by either of two virus variants, Variola major and Variola minor. The disease is also known by the Latin names Variola or Variola vera, derived from varius (“spotted”) or varus (“pimple”). The disease was originally known in English as the “pox” or “red plague”; the term “smallpox” was first used in Britain in the 15th century to distinguish Variola from the “great pox” (syphilis). The last naturally occurring case of smallpox (Variola minor) was diagnosed on 26 Oct. 1977.
Infection with smallpox is focused in small blood vessels of the skin and in the mouth and throat before disseminating. In the skin it results in a characteristic maculopapular rash and, later, raised fluid-filled blisters. V. major produced a more serious disease and had an overall mortality rate of 30-35 percent. V. minor caused a milder form of disease (also known as alastrim, cottonpox, milkpox, whitepox, and Cuban itch) which killed about 1 percent of its victims. Long-term complications of V. major infection included characteristic scars, commonly on the face, which occur in 65-85 percent of survivors. Blindness resulting from corneal ulceration and scarring, and limb deformities due to arthritis and osteomyelitis were less common complications, seen in about 2-5 percent of cases.
Smallpox vaccination within three days of exposure will prevent or significantly lessen the severity of smallpox symptoms in the vast majority of people. Vaccination four to seven days after exposure can offer some protection from disease or may modify the severity of disease. Other than vaccination, treatment of smallpox is primarily supportive, such as wound care and infection control, fluid therapy, and possible ventilator assistance. Flat and hemorrhagic types of smallpox are treated with the same therapies used to treat shock, such as fluid resuscitation. People with semi-confluent and confluent types of smallpox may have therapeutic issues similar to patients with extensive skin burns.
No drug is currently approved for the treatment of smallpox. However, antiviral treatments have improved since the last large smallpox epidemics, and studies suggest that the antiviral drug cidofovir might be useful as a therapeutic agent. The drug must be administered intravenously, however, and may cause serious kidney toxicity.
The overall case-fatality rate for ordinary-type smallpox is about 30 percent, but varies by pock distribution: ordinary type-confluent is fatal about 50-75 percent of the time, ordinary-type semi-confluent about 25-50 percent of the time, in cases where the rash is discrete the case-fatality rate is less than 10 percent. The overall fatality rate for children younger than 1 year of age is 40-50 percent. Hemorrhagic and flat types have the highest fatality rates. The fatality rate for flat-type is 90 percent or greater and nearly 100 percent is observed in cases of hemorrhagic smallpox. The case-fatality rate for Variola minor is 1 percent or less. There is no evidence of chronic or recurrent infection with Variola virus.
In fatal cases of ordinary smallpox, death usually occurs between the tenth and sixteenth days of the illness. The cause of death from smallpox is not clear, but the infection is now known to involve multiple organs. Circulating immune complexes, overwhelming viremia, or an uncontrolled immune response may be contributing factors. In early hemorrhagic smallpox, death occurs suddenly about six days after the fever develops. Cause of death in hemorrhagic cases involved heart failure, sometimes accompanied by pulmonary edema. In late hemorrhagic cases, high and sustained viremia, severe platelet loss and poor immune response were often cited as causes of death. In flat smallpox modes of death are similar to those in burns, with loss of fluid, protein and electrolytes beyond the capacity of the body to replace or acquire, and fulminating sepsis.
Complications of smallpox arise most commonly in the respiratory system and range from simple bronchitis to fatal pneumonia. Respiratory complications tend to develop on about the eighth day of the illness and can be either viral or bacterial in origin. Secondary bacterial infection of the skin is a relatively uncommon complication of smallpox. When this occurs, the fever usually remains elevated.
Other complications include encephalitis (1 in 500 patients), which is more common in adults and may cause temporary disability; permanent pitted scars, most notably on the face; and complications involving the eyes (2 percent of all cases). Pustules can form on the eyelid, conjunctiva, and cornea, leading to complications such as conjunctivitis, keratitis, corneal ulcer, iritis, iridocyclitis, and optic atrophy. Blindness results in approximately 35 percent to 40 percent of eyes affected with keratitis and corneal ulcer. Hemorrhagic smallpox can cause subconjunctival and retinal hemorrhages. In 2 to 5 percent of young children with smallpox, virions reach the joints and bone, causing osteomyelitis variolosa. Lesions are symmetrical, most common in the elbows, tibia, and fibula, and characteristically cause separation of an epiphysis and marked periosteal reactions. Swollen joints limit movement, and arthritis may lead to limb deformities, ankylosis, malformed bones, flail joints, and stubby fingers.
Smallpox is believed to have emerged in human populations about 10,000 BC. The earliest physical evidence of it is probably the pustular rash on the mummified body of Pharaoh Ramses V of Egypt. The disease killed an estimated 400,000 Europeans annually during the closing years of the 18th century (including five reigning monarchs), and was responsible for a third of all blindness. Of all those infected, 20-60 percent—and over 80 percent of infected children—died from the disease. Smallpox was responsible for an estimated 300-500 million deaths during the 20th century. As recently as 1967, the World Health Organization (WHO) estimated that 15 million people contracted the disease and that two million died in that year. After vaccination campaigns throughout the 19th and 20th centuries, the WHO certified the global eradication of smallpox in 1979. Smallpox is one of two infectious diseases to have been eradicated, the other being rinderpest, which was declared eradicated in 2011.
D. Monkeypox
Monkeypox virus (MPV) is a double-stranded DNA, zoonotic virus and a species of the genus Orthopoxvirus in the family Poxviridae. It is one of the human orthopoxviruses that includes Variola (VARY), cowpox (CPX), and vaccinia (VACV) viruses. But it is not a direct ancestor to, nor a direct descendent of, the Variola virus which causes smallpox. The monkeypox virus causes a disease that is similar to smallpox, but with a milder rash and lower death rate. Variation in virulence of the virus has been observed in isolates from Central Africa where strains are more virulent than those from Western Africa.
Monkeypox is carried by both animals and humans. It was first identified by Preben von Magnus in Copenhagen, Denmark in 1958 in crab-eating macaque monkeys (Macaca fascicularis) being used as laboratory animals. It has also been identified in the giant Gambian rat which was the source of a 2003 outbreak in the United States. Monkeypox virus causes the disease in both humans and animals. The crab-eating macaque is often used for neurological experiments. The virus is mainly found in tropical rainforest regions of central and West Africa.
The virus can spread both from animal to human and from human to human. Infection from animal to human can occur via an animal bite or by direct contact with an infected animal's bodily fluids. The virus can spread from human to human by both droplet respiration and contact with fomites from an infected person's bodily fluids. Incubation period is 10-14 days. Prodromal symptoms include swelling of lymph nodes, muscle pain, headache, fever, prior to the emergence of the rash.
The virus is mainly found in the tropical rainforests of Central Africa and West Africa. It was first discovered in monkeys in 1958, and in humans in 1970. Between 1970 and 1986, over 400 cases in humans were reported. Small viral outbreaks with a death rate in the range of 10% and a secondary human to human infection rate of about the same amount occur routinely in equatorial Central and West Africa. The primary route of infection is thought to be contact with the infected animals or their bodily fluids. The first reported outbreak in the United States occurred in 2003 in the midwestern states of Illinois, Indiana, and Wisconsin, with one occurrence in New Jersey. The outbreak was traced to prairie dogs infected from an imported Gambian pouch rat. No deaths occurred.
E. Cowpox
Cowpox is an infectious disease caused by the cowpox virus. The virus, part of the orthopoxvirus family, is closely related to the vaccinia virus. The virus is zoonotic, meaning that it is transferable between species, such as from animal to human. The transferal of the disease was first observed in dairymaids who touched the udders of infected cows and consequently developed the signature pustules on their hands. Cowpox is more commonly found in animals other than bovines, such as rodents. Cowpox is similar to, but much milder than, the highly contagious and often deadly smallpox disease. Its close resemblance to the mild form of smallpox and the observation that dairymaids were immune from smallpox inspired the first smallpox vaccine, created and administered by English physician Edward Jenner.
The word “vaccination,” coined by Jenner in 1796, is derived from the Latin root vaccinus, meaning of or from the cow. Once vaccinated, a patient develops antibodies that make him/her immune to cowpox, but they also develop immunity to the smallpox virus, or Variola virus. The cowpox vaccinations and later incarnations proved so successful that in 1980, the World Health Organization announced that smallpox was the first disease to be eradicated by vaccination efforts worldwide. Other orthopox viruses remain prevalent in certain communities and continue to infect humans, such as the cowpox virus (CPXV) in Europe, vaccinia in Brazil, and monkeypox virus in Central and West Africa.
Today, the virus is found in Europe, mainly in the UK. Human cases are very rare (though in 2010 a laboratory worker contracted cowpox) and most often contracted from domestic cats. Human infections usually remain localized and self-limiting, but can become fatal in immunosuppressed patients. The virus is not commonly found in cattle; the reservoir hosts for the virus are woodland rodents, particularly voles. Domestic cats contract the virus from these rodents. Symptoms in cats include lesions on the face, neck, forelimbs, and paws, and, less commonly, upper respiratory tract infections. Symptoms of infection with cowpox virus in humans are localized, pustular lesions generally found on the hands and limited to the site of introduction. The incubation period is 9 to 10 days. The virus is most prevalent in late summer and autumn.
Immunity to cowpox is gained when the smallpox vaccine is administered. Though the vaccine now uses vaccinia virus, the poxviruses are similar enough that the body becomes immune to both cow- and smallpox.
A. General Methods
It will be understood that monoclonal antibodies binding to Poxvirus will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing Poxvirus infection, as well as for treating the same. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Pat. No. 4,196,265).
The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects that have been exposed to the pathogens, such as those who have been diagnosed as having contracted the disease, or those who have been vaccinated to generate protective immunity against the pathogen. Circulating anti-pathogen antibodies can be detected, and antibody producing B cells from the antibody-positive subject may then be obtained.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.
The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain may also be used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.
Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.
MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.
It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973 which describes antibody-therapeutic agent conjugates.
B. Antibodies of the Present Disclosure
Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. In one aspect, there are provided monoclonal antibodies having clone-paired CDR's from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such antibodies may be produced by the clones discussed below in the Examples section using methods described herein.
In a second aspect, the antibodies may be defined by their variable sequence, which include additional “framework” regions. These are provided in Tables 1 and 2 that encode or represent full variable regions. Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C., (e) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (0 the amino acids may vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing applies to the nucleic acid sequences set forth as Table 1 and the amino acid sequences of Table 2.
C. Engineering of Antibody Sequences
In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. The following is a general discussion of relevant techniques for antibody engineering.
Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.
Recombinant full length IgG antibodies were generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected an purified from the 293 or CHO cell supernatant.
The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.
Antibody molecules will comprise fragments (such as F(ab′), F(ab′)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.
In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (−0.5); acidic amino acids: aspartate (+3.0±1), glutamate (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (−0.4), sulfur containing amino acids: cysteine (−1.0) and methionine (−1.3); hydrophobic, nonaromatic amino acids: valine (−1.5), leucine (−1.8), isoleucine (−1.8), proline (−0.5±1), alanine (−0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (−3.4), phenylalanine (−2.5), and tyrosine (−2.3).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.
Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.
D. Single Chain Antibodies
A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alaine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5×106 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VII C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.
The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.
In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).
Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
U.S. Pat. No. 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
U.S. Pat. No. 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Pat. No. 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.
E. Intrabodies
In a particular embodiment, the antibody is a recombinant antibody that is suitable for action inside of a cell—such antibodies are known as “intrabodies.” These antibodies may interfere with target function by a variety of mechanism, such as by altering intracellular protein trafficking, interfering with enzymatic function, and blocking protein-protein or protein-DNA interactions. In many ways, their structures mimic or parallel those of single chain and single domain antibodies, discussed above. Indeed, single-transcript/single-chain is an important feature that permits intracellular expression in a target cell, and also makes protein transit across cell membranes more feasible. However, additional features are required.
The two major issues impacting the implementation of intrabody therapeutic are delivery, including cell/tissue targeting, and stability. With respect to delivery, a variety of approaches have been employed, such as tissue-directed delivery, use of cell-type specific promoters, viral-based delivery and use of cell-permeability/membrane translocating peptides. With respect to the stability, the approach is generally to either screen by brute force, including methods that involve phage display and may include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement/modification.
An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to subcellular regions such as the cytoplasm, nucleus, mitochondria and ER have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997).
By virtue of their ability to enter cells, intrabodies have additional uses that other types of antibodies may not achieve. In the case of the present antibodies, the ability to interact with the MUC1 cytoplasmic domain in a living cell may interfere with functions associated with the MUC1 CD, such as signaling functions (binding to other molecules) or oligomer formation. In particular, it is contemplated that such antibodies can be used to inhibit MUC1 dimer formation.
F. Purification
In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
A. Formulation and Administration
The present disclosure provides pharmaceutical compositions comprising anti-Poxvirus antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.
Active vaccines are also envisioned where antibodies like those disclosed are produced in vivo in a subject at risk of Poxvirus infection. Such vaccines can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by intradermal and intramuscular routes are contemplated. The vaccine could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts, include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.
Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.
Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as “antibody-directed imaging.” Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.
In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodine123, iodine125, iodine131, indium111, 59iron, 32phosphorus, rhenium186, rhenium188, 75selenium, 35sulphur, technicium99m and/or yttrium90. 125I is often being preferred for use in certain embodiments, and technicium99m and/or indium111 are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure may be labeled with technetium99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraacetic acid (EDTA).
Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
Another type of antibody conjugates contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.
Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.
Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents.
Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxy benzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting Poxvirus and its associated antigens. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of H1 antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate/desired reactivity profiles.
Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of Poxvirus antibodies directed to specific parasite epitopes in samples also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing Poxvirus, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.
These methods include methods for purifying Poxvirus or related antigens from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the Poxvirus or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the Poxvirus antigen immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.
The immunobinding methods also include methods for detecting and quantifying the amount of Poxvirus or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing Poxvirus or its antigens, and contact the sample with an antibody that binds Poxvirus or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing Poxvirus or Poxvirus antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.
Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to Poxvirus or antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.
The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.
Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
A. ELISAs
Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the Poxvirus or Poxvirus antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-Poxvirus antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-Poxvirus antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
In another exemplary ELISA, the samples suspected of containing the Poxvirus or Poxvirus antigen are immobilized onto the well surface and then contacted with the anti-Poxvirus antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-Poxvirus antibodies are detected. Where the initial anti-Poxvirus antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-Poxvirus antibody, with the second antibody being linked to a detectable label.
Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.
“Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25° C. to 27° C., or may be overnight at about 4° C. or so.
Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2′-azino-di-(3-ethyl-benzthiazoline sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.
In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of Poxvirus antibodies in sample. In competition based assays, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample.
Here, the inventors propose the use of labeled Poxvirus monoclonal antibodies to determine the amount of Poxvirus antibodies in a sample. The basic format would include contacting a known amount of Poxvirus monoclonal antibody (linked to a detectable label) with Poxvirus antigen or particle. The Poxvirus antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample.
B. Western Blot
The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.
The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.
C. Immunohistochemistry
The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).
Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in −70° C. isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings. Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.
D. Immunodetection Kits
In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect Poxvirus or Poxvirus antigens, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to Poxvirus or Poxvirus antigen, and optionally an immunodetection reagent.
In certain embodiments, the Poxvirus antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.
Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.
The kits may further comprise a suitably aliquoted composition of the Poxvirus or Poxvirus antigens, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.
The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Donors. PBMCs were obtained from subjects vaccinated with Dryvax (Wyeth), IMVAMUNE (Bavarian Nordic), or ACAM2000 (Acambis). The VRC 201 study was approved by the NIAID IRB under the intramural number 02-1-0316. The ClinicalTrials.gov number was NCT00046397. One sample was obtained from a U.S. survivor of naturally-acquired MPXV infection (Lewis et al., 2007). The studies were approved by the Institutional Review Boards of Vanderbilt University Medical Center, Oregon Health Sciences University, and the National Institute of Allergy and Infectious Diseases.
Mice. C57BL/6 and CBy.Smn.CB17PRKdc SCID/J (BALB/c SCID) mice were purchased from Jackson Laboratories (Bar Harbor). BALB/c SCID mice received Laboratory Autoclavable Rodent Diet #5010 (LabDiet). Breeding, maintenance and experimentation complied with Institutional Animal Care and Use Committee regulations.
Cell lines and viruses. VACV Dryvax (NIH, Lot #4008284), VACV Western Reserve (VACV-WR; ATCC VR-119) and CPXV Brighton Red (BEI Resources, NR-88) were propagated and titered in monolayer cultures of BSC-40 cells (ATCC CRL-2761). MPXV Zaire was propagated in BSC-40 cells and titered on Vero cells (ATCC CCL-81). Bangladesh 1974 Solaiman strain of VARV was propagated in monolayer cultures of Vero E6 cells (ATCC CRL-1586). VACV and CPXV were manipulated under BSL-2 conditions by vaccinated personnel. MPXV was manipulated under BSL-2 conditions with BSL-3 precautions by vaccinated personnel. All experiments with live VARV were reviewed and approved by the World Health Organization Advisory Committee on Variola Virus Research (WHO ACVVR). Experiments with VARV were conducted in accordance with WHO ACVVR guidelines and within a biosafety level 4 laboratory.
Antigens. Recombinant VACV proteins A27, A33, L1, B5, A28, L5, A21, H2, F9, J5, and VARV proteins 12, A31.5, A36, M1, B6, A31 were produced using a baculovirus expression system or purchased from BEI Resources. Truncated monomeric D8 protein was kindly provided by Dr. D. M. Zajonc and Dr. Y. Xiang. Recombinant VACV H3 protein was kindly provided by Dr. Crotty. DNA encoding the MPXV ortholog of the A27 VACV protein was purchased from BEI Resources. H3 and D8 protein orthologs of VARV were produced after WHO approval, as described previously (Davies et al., 2005b; Matho et al., 2012). Cell lysates infected with VACV (NYCBOH), CPXV, MPXV were prepared and inactivated as described previously (Amanna et al., 2012). A VACV-WR protein array was acquired from Antigen Discovery, Inc. The VARV protein microarray was prepared as described previously (Davies et al., 2005b).
Generation of human hybridomas. Human hybridomas were generated as described previously (Crowe, 2009). Briefly, cryopreserved samples were transformed with Epstein-Barr virus. Cultures were incubated in 384-well culture plates for 10 days and then expanded using cell culture medium containing irradiated heterologous human PBMCs (Nashville Red Cross). Plates were screened for VACV recombinant antigen- or VACV-infected cell lysate-specific antibody secreting cell lines using ELISA. Cells from wells with supernatants containing Abs that reacted to antigen or infected cell lysate were fused with HMMA2.5 myeloma cells using an established electrofusion technique (Yu et al., 2008).
ELISA protocol. For screening ELISA, plates were coated with antigen at 1 μg/mL, or 1:400 dilution of a lysate in PBS. After blocking, plates were incubated with culture supernatants followed by incubation with anti-human IgG conjugated with alkaline phosphatase (Meridian, Life Science Inc.) or HRP (Pharmigen). Plates were developed and supernatants were counted as VACV-reactive or recombinant protein antigen-reactive if their absorbance was 2.5-fold above the background from wells containing medium or coated with uninfected cell lysate, respectively. For binding kinetics and cross-reactivity assays, purified mAbs were assessed at concentrations ranging from 100 μg/mL to 20 pg/mL, in triplicate. EC50 values were determined using Prism 5.0 software (GraphPad) after log transformation of antibody concentration using sigmoidal dose-response nonlinear fit analysis with R2 values greater than 0.85, as described previously (Thornburg et al., 2013). Binding of purified mAbs to VARV-infected cell lysate was determined at a single dilution of 100 μg/mL, in triplicate.
MAb isotype analysis. The isotype and subclass of secreted antibodies were determined using murine anti-human IgG1-IgG4 AP-conjugated antibodies (Southern Biotech).
Protein arrays and mAb target analysis. The Orthopoxvirus (VACV strain WR) protein array was acquired from Antigen Discovery, Inc. (ADI). The VARV protein microarray was fabricated in a similar manner as described previously (Davies et al., 2005b). Briefly, individual open reading frames encoded by the viral genome were amplified and cloned into T7 expression vectors by homologous recombination. Proteins were produced using an Escherichia coli-based cell-free coupled transcription/translation reactions (RTS 100 kits; 5 Prime, Gaithersburg, USA) according to the manufacturer's instructions. Proteins were printed without further purification on nitrocellulose-coated glass slides (Whatman). Protein expression was monitored using hemagglutinin or His tags present on the protein termini; quantification of the amount of protein spotted was not possible. No-DNA control spots containing the reaction mixture but lacking template DNA were included throughout the array to correct for background binding to E. coli proteins found in the transcription-translation mixture.
MAbs were probed on the VACV strain WR or VARV protein arrays at dilutions between 1:25 and 1:100, according to the manufacturer's instructions and reagents (ADI). Briefly, arrays were probed with antibody overnight at 4° C., then with biotin-conjugated goat anti-human antibodies for 1 hour at RT, then with a streptavidin-conjugated fluorophore for 1 hour at RT. Arrays were scanned using a GenePix 4100A scanner (Molecular Devices) with laser setting at 100% and photomultiplier (PMT) gain of 400. Image analysis was performed with GenePix Pro 5.0 software (Molecular Devices). Spot intensity was calculated as the median spot value minus local spot background. A secondary correction for background binding to E. coli proteins in the reaction mixture was done by subtracting an average of the no-DNA spots from the background-corrected spot value. Since mAb affinity, protein sequence conservation, and protein expression levels vary, a simple evaluation for highest fluorescent intensity, and a correlation between the two chips, if needed, was used to identify protein targets.
Biolayer interferometry analysis. Experiments were performed on an Octet RED biosensor instrument (Pall ForteBio; Menlo Park) essentially as described previously (Smith et al., 2014). Briefly, biosensors were pre-wetted in running buffer containing DPBS, 0.1% BSA, and 0.05% Tween-20. Human mAbs were loaded onto Protein G biosensor tips (ForteBio) at a concentration of 105 μg/mL and then washed. Biosensors were incubated with a 0.2 mL volume of recombinant protein solution at a 90 μg/mL concentration and washed. Antibody-antigen association/disassociation was determined as wavelength shift in nm.
For competition-binding studies, mAb-antigen complexes were tested for the ability to bind a second mAb in sandwich assay as described previously (Smith et al., 2014). The extent of antibody-antigen association was determined as wavelength shift in nm and calculated as a percentage after normalization, where 0% was the wavelength shift in nm for self-blocking control and 100% was the maximal wavelength shift in nm. Experiments were performed in duplicate. Antibodies were considered to be members of the same competition-binding group if they competed for binding to antigen and exhibited a similar blocking pattern to other antibodies in the panel.
MAb isoype and gene sequence analysis. The isotype and subclass of secreted antibodies were determined using murine anti-human IgG1-IgG4 antibodies followed by secondary anti-mouse HRP-conjugated antibody (Southern Biotech). Nucleotide sequences of variable gene segments were determined by Sanger sequencing from cloned cDNA generated by reverse transcription PCR of mRNA, using variable gene-specific primers designed to amplify antibody genes from all gene families (Weitkamp et al., 2003). Identity of the gene segments and mutations from the germline sequences were determined by alignment using the ImMunoGeneTics database (world-wide-web at imgt.org) (Ruiz et al., 2000).
MAb production and purification. Hybridoma cells secreting VACV-specific mAbs were grown in serum-free medium (Gibco). MAbs were purified from culture supernatants using a HiTrap MabSelect Sure column (GE Healthcare).
Virus neutralization assays. Neutralizing activity of mAbs was determined using MV or EV forms of VACV strain NYCBOH, CPXV, or MPXV, or MV of VARV in a plaque reduction neutralization (PRNT) assay. Neutralization was performed in the presence of complement for all viruses except VARV MV. All experiments with live VARV were reviewed and approved by the World Health Organization Advisory Committee on Variola Virus Research (WHO ACVVR). Experiments with VARV were conducted in accordance with WHO ACVVR guidelines within a biosafety level 4 laboratory. Emax was determined as a maximum of neutralization mAb effect (%); IC50 and Emax values were determined using Prism 5.0 software (GraphPad) after log transformation of antibody concentration using a 3-parameter nonlinear fit analysis of antibody log10 concentration versus response with R2 values greater than 0.85, as described previously (Thornburg et al., 2013).
In vivo protection study. To test the effect of mAbs on respiratory tract infection, six- to eight-week old male B6 mice were injected IP with 100-200 μg of individual mAbs or designated mixtures of mAbs (100-200 μg of each mAb), or 5 mg of VIGIV (BEI Resources). Human anti-dengue virus mAb served as mock-vaccinated control. In ABSL-2 facilities, ketamine-xylazine anesthetized mice were inoculated IN with 105 PFU VACV-WR in 50 μL, or in some experiments in 10 μL of PBS. In some experiments, mice were inoculated with 106 PFU VACV. For virus titer determination, lungs from individual mice were homogenized and plated on confluent BSC-40 cell monolayer cultures. To test the effect of mAbs on disseminated VACV infection, eight- to ten-week old female BALB/c SCID mice were given Abs IP either prior to or after VACV inoculation, as detailed in the text. For lethal challenge, mice were inoculated IP with 105 PFU VACV-WR in 100 μL PBS. Mice were weighed and monitored daily for morbidity, and those losing over 30% of initial body weight were euthanized, per IACUC requirements.
Quantification and Statistical Analysis. The descriptive statistics mean±SEM or mean±SD were determined for continuous variables as noted. Comparisons were performed using Wilcoxon rank sum test or the post hoc group comparisons in ANOVA; all tests were two-tailed and unpaired. Survival curves were estimated using the Kaplan Meier method and curves compared using the log rank test with subjects right censored, if they survived until the end of the study. * −p<0.05; ** − was used to reject a “null hypothesis”. *=p<0.05; **=p<0.01; *** −=p<0.001; ns—non-significant. Statistical analyses were performed using Prism v5.0 (GraphPad).
Poxvirus infection in humans elicits a complex B cell response encoding large numbers of clones reactive with antigens from diverse Orthopoxvirus species. The inventors obtained peripheral blood mononuclear cells (PBMCs) from a donor who had recovered from a naturally-occurring MPXV infection or from otherwise healthy subjects previously immunized with one of three different vaccine formulations (Table S1), IMVAMUNE (live attenuated modified vaccinia Ankara virus), Dryvax (a freeze-dried calf lymph produced vaccinia virus), or ACAM2000 (Vero cell culture produced vaccinia virus) (Verardi et al., 2012). To identify poxvirus-specific B cell cultures, PBMCs were transformed with Epstein-Barr virus, and the supernatants from the resulting lymphoblastoid cell lines were screened by ELISA for binding to poxvirus antigens. Hybridomas secreting human antigen-specific mAbs were generated from B cell lines secreting virus-specific antibodies, as previously described (Crowe, 2009). For screening, the inventors used 12 recombinant VACV proteins antigens designated A21, A27, A28, A33, B5, D8, F9, J5, H2, H3, L1, and L5. The A33 and B5 proteins are surface antigens on the EV form of virus, while the remaining ten proteins are surface antigens on MV particles. The inventors also screened supernatants for binding to inactivated lysates of VACV-infected BSC-40 cell monolayer cultures.
A total of 89 cloned hybridoma cell lines secreting human mAbs were isolated, including 44 lines from vaccinees and 45 from the donor with a history of MPXV infection (Table S1). The 89 mAbs were independent clones that displayed a high degree of sequence diversity, including a unique HCDR3 sequence for each mAb (Table S2). Thirty-two mAbs in the panel bound in ELISA to inactivated VACV-infected cell lysates only, and thus their protein antigen specificity was uncertain initially. Binding of these mAbs was reassessed using VACV protein antigen microarrays, which revealed additional mAbs specific to D8, H3 A21, A25, H5 and I1 VACV proteins. Therefore, the mAb panel contained Abs to at least 12 antigens: D8, B5, A33, H3, L1, A27, I1, A25, F9, A28, A21, and H5 (
The inventors next assessed the cross-reactivity of individual VACV-reactive mAbs to CPXV, MPXV or VARV by testing binding to CPXV-, MPXV- or VARV-infected cell lysates or to recombinant VARV protein antigens that are orthologs of the identified VACV targets. A large fraction (45 of 73-62%) of mAbs that bound to VACV antigens in virus-infected cell lysate (
The majority of human neutralizing mAbs recognized one of six antigens and exhibited cross-neutralization for several Orthopoxvirus species. The inventors next tested the mAbs in virus neutralization assays using MV or EV forms of VACV, CPXV, or MPXV. Neutralization potency of mAbs was assessed based on the half-maximal inhibitory concentration (IC50) and the maximum of neutralization effect (Emax) values. More than half (48 of 89-54%) of the mAbs possessed neutralizing activity (Emax≥50%) at 100 μg/mL or lower concentration for at least one orthopoxvirus; 16 or 32 mAbs neutralized the EV or MV form of VACV, respectively (
A majority (38 of 48-79%) of neutralizing mAbs cross-neutralized at least two Orthopoxvirus species (mainly VACV and CPXV), and 12 of 48 (25%) mAbs neutralized three orthopoxviruses—VACV, CPXV and MPXV (
Mixtures of diverse mAb specificities possess superior cross-neutralizing activity for VACV, CPXV, MPXV, and VARV. The inventors next designed two mixtures of mAbs, designated M
Superior in vivo protection against VACV infection was achieved by administration of a mixture of human mAbs that targeted multiple viral antigens. The inventors next evaluated the protective capacity of M
To further characterize the protective efficacy of M
In summary, these findings demonstrate the high prophylactic potency of M
Four principal antibody specificities participated in protection against respiratory VACV challenge when used in mixture. The inventors next determined the contribution of individual mAbs within M
It was possible that some of the six Ab specificities contributed to protection in mixtures only in a cooperative manner that would not be detected by monotherapy studies. To detect such activity, the inventors designed mixtures that were variants of the M
One possible explanation for the diminished protection observed when a mAb mixture lacked a single MV- or EV-targeted mAb specificity was the decrease in total amount of mAb per treatment. Therefore, the inventors next examined whether the lack of one mAb specificity in protective M
Therapeutic effect of M
Diverse human Ab specificities participate in protection against systemic VACV infection. The experiments described above showed that a single dose of M
In this study, the inventors elucidated the breadth and specificity of human cross-neutralizing mAbs against the clinically relevant orthopoxviruses VACV, CPXV, MPXV, and VARV. In addition, they identified protective specificities for human mAbs and demonstrated that superior protection in mouse challenge models could be achieved with a defined mAb mixture that targeted a limited number of poxvirus protein antigens.
Studying protective antibody-mediated immunity for poxvirus infections has been challenging because of the lack of clonal human Abs representing the naturally-occurring human B cell response to poxvirus infection or immunization. In the current work, using a cohort of orthopoxvirus-immune subjects, the inventors showed that orthopoxvirus infection elicits a complex B cell response encoding large numbers of clones reactive to antigens from diverse orthopoxvirus species. Further analysis of individual clones revealed the importance of six major neutralizing mAb specificities that targeted both MV (anti-H3, -A27, -D8 and -L1) and EV (anti-B5 and -A33) infectious forms of poxvirus and required complement for optimal activity.
In studies of human mAbs to other viruses, such as HIV, influenza, or dengue virus, the inventors have found that the percentage of neutralizing mAbs among the total number of mAbs induced by infection or vaccination varies according to the agent. For example, typically less than 1% of the mAbs induced by dengue virus infection neutralize virus (Smith et al., 2012), whereas a large proportion of influenza-specific mAbs neutralize (Thornburg et al., 2013). For orthopoxviruses, the inventors found here that a high fraction of the mAbs from the panel (54%) possessed neutralizing activity. Given the high level of sequence homology among the surface proteins from VACV, CPXV, MPXV and VARV (89-100%), such a robust and diverse neutralizing Ab response likely explains the efficient cross-protection induced by VACV immunization against heterologous orthopoxvirus infections. The inventors' finding that a large fraction of poxvirus-specific mAbs of the panel exhibited cross-binding and/or cross-neutralizing activity for VACV, CPXV, MPXV, and VARV further substantiates this model. The broadest cross-neutralization was achieved by mAbs targeting four antigens in the MV form of VACV, namely A33, A27, L1 and H3 (or the ortholog proteins in the other three viruses), thus identifying the principal determinants of Ab-mediated cross-protective immunity to orthopoxviruses. Of note, the presence of complement enhanced the inhibitory activity of mAbs targeting most neutralizing determinants.
Information about the protective potential of human Abs has been limited mostly to the study of varying lots of VIGIV, which has been used with partial success for post-exposure treatment and for management of some severe adverse reactions to smallpox vaccination (Wittek, 2006). Multiple antigen specificities appear to contribute to neutralization of the MV form of VACV by VIGIV or immune serum IgG (Benhnia et al., 2008; Moss, 2011). Abs to B5 were thought responsible for much of the neutralization activity against VACV EV forms of virus (Bell et al., 2004). Animal studies suggested that protection is not readily achieved by administration of a single neutralizing mAb, and requires both EV- and MV-targeted mAbs (Lustig et al., 2005). Reconstituting (or improving) the protective activity of VIGIV with mAbs has been attempted empirically, using a mixture of anti-H3 and -B5 mAbs (McCausland et al., 2010), or a complex mixture of 26 human mAbs directed to fourteen antigens (Lantto et al., 2011; Zaitseva et al., 2011). These data suggest that a mixture containing mAbs of only two specificities (anti-H3 and anti-B5) likely would fail to cross-protect efficiently, since the inventors observed that anti-B5 mAbs fail to neutralize the EV form of MPXV. In contrast, the previous mixture of 26 mAbs likely includes redundant or noncontributory mAbs, because this composition contains a number of mAbs that are directed to antigenic specificities without an apparent role in cross-neutralization or protection. To make a potent neutralizing and protective human Ab mixture by rational design that recognizes the four major poxvirus threats to humans, the inventors combined potent cross-neutralizing human mAbs targeting six major poxvirus antigenic proteins: the MV antigens H3, A27, D8 and L1 and the EV antigens B5 and A33. Remarkably, M
Poxviruses transmit by several routes of infection, and cause diverse clinical syndromes in humans (Smith and McFadden, 2002), which can be modeled in part using different animal models (Chapman et al., 2010). The inventors sought here to compare the prophylactic and treatment efficiency of human mAbs and their mixtures in several well-established VACV lethal challenge murine models using either mild or severe respiratory tract infection or, alternatively, systemic inoculation resulting in disseminated infection (Belyakov et al., 2003; Flexner et al., 1987; Wyatt et al., 2004). The resulting data revealed that the contribution of individual specificities to protection varied depending on the route of virus inoculation. Four specificities (anti-A33, -B5, -L1 and -A27), contributed significantly to protection against respiratory tract infection, while in contrast, all six tested specificities contributed to protection in the model of systemic infection. Moreover, the inventors observed that the major contribution to protection in both models was provided by EV-targeted anti-B5 and -A33 human mAbs, consistent with previous studies of mouse mAbs (Lustig et al., 2005). Thus, cross-protection against all clinically important orthopoxviruses is most likely achieved when incorporating both EV-neutralizing anti-B5 and -A33 mAbs, which may compensate for some species cross-neutralization deficiencies of the other. M
Using naturally occurring human mAbs isolated using hybridoma technology, this study revealed six principal cross-neutralizing human mAb specificities for VACV, CPXV, MPXV and VARV, and Ab specificities that are necessary and sufficient determinants of protection in murine challenge models. This work suggests that a mixture of these Abs could mediate cross-protective immunity to orthopoxviruses. As with most studies, there are several limitations of this work that the inventors would like to point out. First, the antibody discovery platform used likely allowed us to identify mAbs only from the most frequent classes of B cell memory clones that occur in human peripheral blood. Therefore, less frequent clones could be missing from the inventors' analysis. Second, it remains unknown to what extent the B cell memory repertoire in the blood that the inventors have studied corresponds to the antigen-reactive antibody protein repertoire in the serum that is secreted by long-lived plasma cells in the bone marrow. Future proteomics studies using emerging technologies might be able to address this question. And third, future development for use in humans of individual mAbs or mixtures described here against VACV, CPXV, and MPXV or VARV should include studies of larger animal models, such as non-human primates.
aBased on plaque reduction neutralizing test performed using VACV strain WR.
aRange of mAb binding efficiency to VARV-infected cell lysate, where numbers indicate optical density from ELISA:
bMAbs with low expression that were excluded from the analysis
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2017/057150, filed Oct. 18, 2017, which claims benefit of priority to U.S. Provisional Application Ser. No. 62/410,207, filed Oct. 19, 2016, the entire contents of each of which is hereby incorporated by reference.
This invention was made with government support under grant number HHSN272200900047C awarded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/057150 | 10/18/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/075621 | 4/26/2018 | WO | A |
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